Method for nucleic acid transfection of cells

ABSTRACT

The present invention describes methods for introducing nucleic acids into a target cell using a transition metal enhancer. A mixture containing nucleic acid and a transition metal enhancer is exposed to cells. The nucleic acid is taken up into the interior of the cell with the aid of the transition metal enhancer. Since nucleic acids can encode a gene, the method can be used to replace a missing or defective gene in the cell. The method can also be used to deliver exogenous nucleic acids operatively coding for proteins that are secreted or released from target cells, thus resulting in a desired biological effect outside the cell. Alternatively, the methods of the present invention can be used to deliver exogenous nucleic acids into a target cell that are capable of regulating the expression of a predetermined endogenous gene. This can be accomplished by encoding the predetermined endogenous gene on the nucleic acid or by encoding the nucleic acid with a sequence that is the Watson-Crick complement of the mRNA corresponding to the endogenous gene.

1. BRIEF DESCRIPTION OF THE INVENTION

[0001] This application is a continuation-in-part of co-pendingapplication Ser. No. 09/487,089 filed Jan. 19, 2000. The presentinvention relates to methods for the delivery of a nucleic acid into acell. The nucleic acid is delivered in combination with a transitionmetal enhancer, which acts as an enhancing agent for effective nucleicacid delivery into a cell, thereby effecting a desired physiologicalconsequence, such as expression of an exogenous protein encoded by thenucleic acid. In some embodiments the nucleic acid is combined withtransition metal enhancer as well as a cationic lipid in order todeliver a nucleic acid into a cell.

2. BACKGROUND OF THE INVENTION

[0002] The advent of recombinant DNA technology and genetic engineeringhas led to numerous efforts to develop methods that facilitate thetransfection of therapeutic and other nucleic acid-based agents tospecific cells and tissues. Known techniques provide for the delivery ofsuch agents, including a variety of genes that are carried inrecombinant expression constructs. These constructs are capable ofmediating expression of the genes once they arrive within a cell. Suchdevelopments have been critical to many forms of molecular medicine,specifically gene therapy, whereby a missing or defective gene can bereplaced by an exogenous copy of the functional gene.

[0003] Typically, nucleic acids are large, highly polar molecules. Assuch, nucleic acids face the impermeable barrier of the cellularmembrane in eukaryotes and prokaryotes. The cell membrane acts to limitor prevent the entry of the nucleic acid into the cell. The developmentof various gene delivery methods has paralleled currently known genetherapy protocols. While much progress has been made in increasing theefficiency of gene delivery into cells, limited nucleic acid uptake ortransfection remains a hindrance to the development of efficient genetherapy techniques.

[0004] Common approaches for delivering a nucleic acid into a cellinclude ex vivo and in vivo strategies. In ex vivo gene therapy methods,the cells are removed from the host organism, such as a human, prior toexperimental manipulation. These cells are then transfected with anucleic acid in vitro using methods well known in the art. Thesegenetically manipulated cells are then reintroduced into the hostorganism. Alternatively, in vivo gene therapy approaches do not requireremoval of the target cells from the host organism. Rather, the nucleicacid may be complexed with reagents, such as liposomes or retroviruses,and subsequently administered to target cells within the organism usingknown methods. See, e.g., Morgan et al., Science 237:1476, 1987; Gerrardet al., Nat. Genet. 3:180, 1993.

[0005] Several different methods for transfecting cells can be used foreither ex vivo or in vivo gene therapy approaches. Known transfectionmethods may be classified according to the agent used to deliver aselect nucleic acid into the target cell. These transfection agentsinclude virus dependent, lipid dependent, peptide dependent, and directtransfection (“naked DNA”) approaches. Other approaches used fortransfection include calcium co-precipitation and electroporation.

[0006] Viral approaches use a genetically engineered virus to infect ahost cell, thereby “transfecting” the cell with an exogenous nucleicacid. Among known viral vectors are recombinant viruses, of whichexamples have been disclosed, including poxviruses, herpesviruses,adenoviruses, and retroviruses. Such recombinants can carry heterologousgenes under the control of promoters or enhancer elements, and are ableto cause their expression in vector-infected host cells. Recombinantviruses of the vaccinia and other types are reviewed by Mackett et al.,J. Virol. 49:3, 1994; also see Kotani et al., Hum. Gene Ther. 5:19,1994.

[0007] However, viral transfection approaches carry a risk ofmutagenicity due to possible viral integration into the cellular genome,or as a result of undesirable viral propagation. Many studies invertebrate systems have established that insertion of retroviral DNA canresult in inactivation or ectopic activation of cellular genes, therebycausing diseases. For a review, see Lee et al., J. Virol. 64:5958-5965,1990. For example, one well known consequence of retroviral integrationis activation of oncogenes. One study describes the activation of ahuman oncogene by insertion of HIV. Shiramizu et al., Cancer Res.,54:2069-2072, 1994. Viral vectors also are susceptible to interferencefrom the host immune system.

[0008] Non-viral vectors, such as liposomes, may also be used asvehicles for nucleic acid delivery in gene therapy. In comparison toviral vectors, liposomes are safer, have higher capacity, are lesstoxic, can deliver a variety of nucleic acid-based molecules, and arerelatively nonimmunogenic. See Felgner, P. L. and Ringold, G. M., Nature337, 387-388, 1989. Among these vectors, cationic liposomes are the moststudied due to their effectiveness in mediating mammalian celltransfection in vitro. One technique, known as lipofection, uses alipoplex made of a nucleic acid and a cationic lipid that facilitatestransfection into cells. The lipid/nucleic acid complex fuses orotherwise disrupts the plasma or endosomal membranes and transfers thenucleic acid into cells. Lipofection is typically more efficient inintroducing DNA into cells than calcium phosphate transfection methods.Chang et al., Focus 10:66, 1988. However, some of the lipid complexescommonly used with lipofection techniques are cytotoxic or haveundesirable non-specific interactions with charged serum components,blood cells, and the extracellular matrix. Furthermore, these liposomecomplexes can promote excessive non-specific tissue uptake.

[0009] One known protein dependent approach involves the use ofpolylysine mixed with a nucleic acid. The polysine/nucleic acid complexis then exposed to target cells for entry. See, e.g., Verma and Somia,Nature 389:239, 1997; Wolffet al., Science 247:1465, 1990. However,protein dependent approaches are disadvantageous because they aregenerally not effective and typically require chaotropic concentrationsof polylysine.

[0010] “Naked” DNA transfection approaches involve methods where nucleicacids are administered directly in vivo. See U.S. Pat. No. 5,837,693 toGerman et al. Administration of the nucleic acid could be by injectioninto the interstitial space of tissues in organs, such as muscle orskin, introduction directly into the bloodstream, into desirable bodycavities, or, alternatively, by inhalation. In these “Naked” DNAapproaches, the nucleic acid is injected or otherwise contacted with theanimal without any adjuvants, such as lipids or proteins, whichtypically results in only moderate levels of transfection, and theinsufficient expression of the desired protein product. It has recentlybeen reported that injection of free (“naked”) plasmid DNA directly intobody tissues, such as skeletal muscle or skin, can lead to proteinexpression, but also to the induction of cytotoxic T lymphocytes andantibodies against the encoded protein antigens contained in theplasmid. See Ulmer et al., Science, 259, 1993, 1745-1749; Wang et al.,Proc. Nat. Acad. Sci. U.S.A. 90, 4157-4160, 1993; Raz et al., Proc. Nat.Acad. Sci. U.S.A. 91, 9519-9523, 1994.

[0011] Electroporation is another transfection method. See U.S. Pat.Nos. 4,394,448 to Szoka, Jr., et al. and U.S. Pat. No. 4,619,794 toHauser. The application of brief, high-voltage electric pulses to avariety of animal and plant cells leads to the formation ofnanometer-sized pores in the plasma membrane. DNA can enter directlyinto the cell cytoplasm either through these small pores or as aconsequence of the redistribution of membrane components thataccompanies closure of the pores. The use of electroporation as a toolto deliver DNA into cells has had limited success for in vivoapplications.

[0012] A common disadvantage to known non-viral nucleic acid deliverytechniques is that the amount of exogenous protein expression producedrelative to the amount of exogenous nucleic acid administered remainstoo low for most diagnostic or therapeutic procedures. Low levels ofprotein expression are often a result of a low rate of transfection ofthe nucleic acid or the instability of the nucleic acid.

[0013] Despite numerous research efforts directed at finding efficientmethods for nucleic acid delivery, most known techniques fail to resultin sufficient cell transfection to achieve the desired proteinexpression. There is still a need to develop a nucleic acid deliverymethod that efficiently introduces recombinant expression constructsencoding useful genes into cells, while minimizing undesirable effects.

3. SUMMARY OF THE INVENTION

[0014] The present invention describes methods for introducing nucleicacids into a target cell using a transition metal enhancer. Inaccordance with the methods of the present invention, a mixturecontaining the nucleic acid and a transition metal enhancer is exposedto cells. The nucleic acid is then taken up into the interior of thecell with the aid of the transition metal enhancer. Since nucleic acidscan encode a gene, the method can be used to replace a missing ordefective gene in the cell. The method can also be used to deliverexogenous nucleic acids operatively coding for polypeptides that aresecreted or released from target cells, thus resulting in a desiredbiological effect outside the cell. Alternatively, the methods can beused to deliver exogenous nucleic acids into a target cell that arecapable of regulating the expression of a predetermined endogenous gene.This can be accomplished by encoding the predetermined endogenous geneon the nucleic acid or by encoding the nucleic acid with a sequence thatis the Watson-Crick complement of the mRNA corresponding to theendogenous gene.

[0015] In particular, the present invention relates to a method fordelivering a nucleic acid into a target cell by contacting a cell with asolution containing a nucleic acid and a transition metal enhancer. Thecell may be derived from or contained within an organism or a primarycell culture. The nucleic acid sequence to be delivered is normallydetermined prior to use of the disclosed method. In some embodiments, anucleic acid is delivered to a target cell by contacting a cell with asolution containing a nucleic acid, a transition metal enhancer andcationic lipid.

[0016] In one embodiment, the solution that facilitates intracellulardelivery of therapeutically effective amounts of nucleic acid to targetcells may be suitable for use with a variety of cell types including,but not limited to, those associated with the various secretory glands(e.g., mammary, thyroid, pancreas, stomach, and salivary glands),musculature connective tissue, bone, bladder, skin, liver, lung, kidney,the various reproductive organs such as testes, uterus and ovaries,nervous system, all other epithelial, endothelial, and mesodermaltissues.

[0017] In other embodiments, the transition metal enhancer is a complex,adduct, cluster or salt of a d-block element, a lanthanide, aluminum,and/or gallium. In yet other embodiments, the transition metal enhanceris a zinc, nickel, cobalt, copper, aluminum, or gallium complex.

[0018] The present invention provides a novel method for delivering anucleic acid into a target cell. In accordance with the methods of thepresent invention, the nucleic acid and transition metal enhancer areexposed to cells. When the nucleic acid encodes a useful protein, theexposure may result in measurable expression of the protein. Suchprotein expression is useful in the practice of both diagnostic andtherapeutic strategies.

4. BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 is a schematic view of the recombinant plasmidpCMV.FOX.Luc-2, which encodes the luciferase gene.

[0020]FIG. 2 is a schematic view of the recombinant plasmid pBAT-iMG-2,which encodes the human alpha-1 antitrypsin gene.

[0021]FIG. 3 is a chart showing the results of an experiment in whichcationic lipid/pCMV.FOX.Luc.2 complexes at various charge ratios werescreened for transfection activity in NIH 3T3 cells in the presence ofvarious concentrations of ZnCl₂.

5. DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention provides a method for transfection of anucleic acid into a cell using a transition metal enhancer. Inparticular, a method for delivering a recombinant expression constructencoding a functional nucleic acid in the presence of a transition metalenhancer is disclosed. For the purposes of this invention, the term“recombinant expression construct” as used herein, is intended to mean anucleic acid encoding a gene or fragment thereof, operably linked to asuitable control sequence capable of effecting the expression of thegene in a suitable host cell. Expressly intended to fall within thedefinition of a “gene” are embodiments comprising cDNA and genomic DNAencoding eukaryotic genes, as well as chimeric hybrids thereof. Alsointended to fall within the scope of the recombinant expressionconstructs of the invention are fragments of such genes which, whenexpressed, may inhibit or suppress the function of an endogenous gene ina cell, including, antisense gene fragments.

[0023] The present invention describes a method for the delivery of anexogenous nucleic acid into a cell in the presence of a transition metalenhancer. The terms “delivery” or “deliver”, as used in reference tonucleic acids, means that a nucleic acid and a cell are brought togethersuch that the nucleic acid may contact and enter the cell. Nucleic aciddelivery according to the methods of the present invention means thatthe nucleic acid comes into contact with a cell in the presence of atransition metal enhancer.

[0024] The entry of a nucleic acid into a cell using the methods ofnucleic acid delivery of the present invention may take place in any wayand preferably leads to an increase in the amount of the nucleic acid inthe cell. Moreover, a nucleic acid delivered into a cell using themethods of the present invention is present in an active form within thecell, i.e., it is capable of being transcribed, or may be capable ofhybridizing to other nucleic acids, or it is capable of being translatedinto a functional protein product.

[0025] Nucleic acids of any kind may be delivered into a cell,including, but not limited to, naturally occurring nucleic acids (e.g.,genomic DNA, mRNA, tRNA, etc.), any synthetic nucleic acid, nucleicacids that have been modified, and nucleic acids that include one ormore protecting groups. The nucleic acids may be delivered to the targetcells using various in vivo, ex vivo or in vitro techniques.

[0026] In one embodiment, nucleic acids that can be used in accordancewith the present invention include genomic or cDNA nucleic acids wellknown in the art. Typically, nucleic acid sequence information for adesired protein can be found in one of many public databases, such as,for example, GENBANK, EMBL, Swiss-Prot. Nucleic acid sequenceinformation may also be found in journal publications. Thus, one ofskill in the art has access to nucleic acid information for virtuallyall known genes having a published sequence. Therefore, in accordancewith the present invention, one of skill in the art can either obtainthe corresponding nucleic acid molecule directly from a publicdepository, or the institution or researcher that published thesequence.

[0027] In another embodiment, the cDNA encoding the desired proteinproduct can then be used to make nucleic acid expression constructs andvectors as described herein. See, e.g., Vallette et al., 1989, NucleicAcids Res., 17:723-733; and Yon and Fried, 1989, Nucleic Acids Res.,17:4895. Thus, virtually all known nucleic acids encoding a therapeuticnucleic acid sequence of interest are appropriate for use in the methodsof the present invention.

[0028] Nucleic acid delivery according to the methods of the presentinvention discloses that a nucleic acid, a transition metal enhancer,and a target cell are brought together sequentially or collectively, asin a solution. In this way, the nucleic acid and the transition metalenhancer are allowed to contact each other prior to contact with thetarget cell. The mixing or bringing together of the nucleic acid, thetransition metal enhancer and the target cell can be accomplished in anyway known to the skilled person in the art.

[0029] In the methods of the present invention, nucleic acid/transitionmetal enhancer mixture can be formed by mixing an exogenous nucleic acidof interest with at least one transition metal enhancer. The nucleicacid/transition metal enhancer mixture is then administered to targetcells. “Administration” may be defined as any route that will exposenucleic acids to target cells. For example, the solution may beadministered intramuscularly, intratracheally, intraperitoneally,intradermally, intravenously, intraperineally, subcutaneously,intraductally, sublingually, by intranasal inhalation, intranasalinstillation, intrarectally, intravaginally, ocularly, orally,intraductally into the ducts of the exocrine glands, and/or topical genedelivery. Examples of target cells that can receive the nucleicacid/transition metal enhancer mixture are an exocrine gland. An“exocrine gland” can be defined as a gland that releases a secretionexternal to or at the surface of an organ by means of a duct or a canal.Examples of exocrine glands are a salivary gland and the pancreas.Alternatively, target cells may be collected from the organism ofinterest and used to establish a primary culture using methods known inthe art. The primary culture may then be contacted with the nucleicacid/transition metal enhancer mixture to allow physical uptake of thenucleic acid by cells of the primary culture. Then, the cells may bereintroduced to the target organism.

[0030] The present invention may be used in accordance with known invivo and/or ex vivo gene therapy methods. For example, when the nucleicacid/transition metal enhancer mixture is used in ex vivo gene therapytechniques, target cells are collected from the organism of interest andthen exposed directly to the nucleic acid/transition metal enhancermixture. Alternatively, when the nucleic acid/transition metal enhancersolution is applied to various desirable in vivo approaches, the nucleicacid/transition metal enhancer mixture may be directly exposed to targetcells following administration. For example, the target cells may beexposed to the gene by injecting the nucleic acid/transition metalenhancer solution into the interstitial space of tissues containing thetarget cells. More specifically, when the target cells of interest aremuscle or skin cells, the nucleic acid/transition metal enhancer mixturemay be injected into the interstitial space of muscle or skin. Inaddition to known applications in gene therapy, the methods of thepresent invention may be novelly applied as a general method in anyapplication that requires physical uptake of nucleic acids into cells.

[0031] The application of the method of the present invention as appliedto in vivo and ex vivo gene therapy approaches merely serves toillustrate one embodiment for the methods described by the presentinvention. In fact, target cells may be exposed to the nucleicacid/transition metal enhancer solution by any conventional techniquebeyond those typically used in in vivo and ex vivo gene therapyapproaches.

[0032] Regardless of any method known to be in vivo, ex vivo, or anyother method used to expose the nucleic acid/transition metal enhancerto target cells, sufficient exposure of the solution to the target cellswill allow for the physical uptake of the nucleic acid into the targetcells. In a preferred embodiment, the exogenous nucleic acid of interestcodes for a polypeptide and is operably linked to a desired promoterthat can cause transfection in the target cells. As defined herein,stable transfection occurs when the exogenous nucleic acid of interestis successfully incorporated into the genome of the target cell.Transient transfection is defined herein as any type of transfectionthat does not rely on the incorporation of the exogenous nucleic acidinto the genome of the target cell.

[0033] In one embodiment, in which a portion of the exogenous nucleicacid of interest is transcribed, the transcribed mRNA is translated intoa protein of interest. The translated protein may have a desiredbiological effect within the target cell, or alternatively, the targetedcell may secrete or release the translated protein and the protein maymanifest a desired biological effect outside the cell.

5.1 Transition Metal Enhancers Useful For Nucleic Acid Delivery 5.1.1General Description of Useful Transition Metal Enhancers

[0034] The transition metal enhancers of the present invention includetransition metals, transition metal complexes, transition metal adducts,transition metal clusters, transition metal salts, and mixtures thereof.The transition metal enhancers also include any transition metalexisting in chemical combination with a variety of other elements in avariety of ways. Specifically, transition metal atoms in the transitionmetal enhancers of the present invention may exist in one or moreoxidation states, i.e., as a free ion or in bound form. In thetransition metal enhancers of the present invention, transition metalatoms may themselves be directly bonded to ligands in complexes, looselyassociated with other chemical species in adducts, or as ions in directcontact with other ions of opposite charge, “counter ions,” or in salts.Complexes may have an overall charge and consequently be associated withcounter-ions, to maintain neutrality.

[0035] The transition metal enhancers of the present invention includecompounds having one or more transition metal atoms selected from theelements in Groups IIIB, IVB, VB, VIIB, VIIIB, IB, and IIB of theperiodic table. This group of elements is defined herein as the d-block.See, e.g., Huheey, INORGANIC CHEMISTRY, Harper & Row, New York, 1983.The transition metal enhancers of the present invention also includethose lanthanides and main group elements having chemical propertiessimilar to transition metal complexes. As defined herein, lanthanidesare the first row of the f-block of the periodic table and main groupelements are those in groups IIIA, IVA, VA and VIIA of the periodictable, the first five groups of which is known to those of skill in theart as the p-block.

[0036] The transition metal enhancers of the present invention may befound in any complex form having any coordination number that ischemically possible, including, but not limited to a coordination numberof 1, 2, 3, 4, 5, 6, 7, 8, or higher, and may further exhibit anygeometric arrangement of ligands about the transition metal atom or ionincluding, but not limited to, tetrahedral, octahedral, square planar,trigonal bipyramidal, square based 10pyramidal, pentagonal bipyramidaland cubic. Furthermore, for any given shape, the transition metalenhancers of the present invention may exhibit any permittedstereochemistry, including, but not limited to cis and trans isomerismand may also undergo fluxional behavior whereby different isomersinterchange faster than the timescale of observation. Furthermore, thetransition metals enhancers of the present invention may be in anychemically possible oxidation state including, but not limited to,oxidation states zero, one, two, three or four and those that areformally negative. In addition, the present invention includes anyisotope of any of the transition metals. The transition metal enhancersof the present invention may also include the transition metal atom oran ion free of any ligands.

5.1.2 Transition Metals

[0037] Any of the following metals may be combined with any inorganic ororganic ligands, or mixtures of such ligands, to form the transitionmetal enhancer according to the methods of the present invention.

5.1.2.1 d-Block Elements

[0038] The transition metal enhancer of the present invention, includecompounds containing scandium, titanium, vanadium, chromium, manganese,iron, cobalt, nickel, copper, zinc, yttrium, zirconium, niobium,molybdenum, technetium, ruthenium, rhodium, palladium, silver, cadmium,lanthanum, hafnium, tantalum, tungsten, rhenium, osmium, iridium,platinum, gold, mercury, or actinium. The transition metal enhancers ofthe present invention may also include compounds derived from membersderived from the d-block commonly categorized as “trans-Actinide”elements, including rutherfordium, hahnium, and elements having anatomic number between 106 and 112.

5.1.2.2 Lanthanides

[0039] The transition metal enhancers of the present invention may alsoinclude lanthanide metals, complexes, adducts, clusters, salts, orenhancers thereof. As defined herein, the lanthanides include cerium,samarium, and gadolinium.

5.1.2.3 p-block Elements

[0040] The transition metal enhancers of the present invention includecomplexes, adducts, clusters, salts, and mixtures thereof, including ap-block element that has properties like transition metals such ascopper. Therefore, the transition metal enhancers of the presentinvention include complexes, adducts, clusters, and/or salts thatinclude aluminum, gallium, indium, tin, antimony, thallium, and lead.

5.1.3 Transition Metal Ligands

[0041] The ligands that may be used to complex or form adducts with thetransition metals, and the similar members from the lanthanide seriesand p-block elements, to form a transition metal enhancer used accordingto the present invention, may be taken from the set of inorganicreagents as well as classes of compounds commonly found in organicchemistry.

5.1.3.1 Inorganic Ligands

[0042] The inorganic reagents that may be used to complex the elementscomprising the transition metals to form the transition metal enhancersof the present invention include, but are not limited to, ammonia,cyanide anion, halides (including bromide, chloride, fluoride, andiodide), hydroxide, dinitrogen, carbon monoxide, dioxygen, oxychloride,hydrogen, water, and mixtures thereof.

5.1.3.2 Organic Ligands

[0043] The compounds that may be used to complex transition metals toform the transition metal enhancers of the present invention include,but are not limited to, alkyls, substituted alkyls, alkenyls,substituted alkenyls, cycloalkyls, substituted cycloalkyls,heterocycloalkyls, substituted heterocycloalkyls, aryls, alkaryls,heteroaryls, and alkheteroaryls.

[0044] As defined herein, alkyls are saturated branched, straight chainor cyclic hydrocarbon radicals. Typical alkyl groups include, but arenot limited to, methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl,isobutyl, t-butyl, cyclobutyl, pentyl, isopentyl, cyclopentyl, hexyl,cyclohexyl and the like. In a preferred embodiment, the alkyl groups ofthe present invention are (C₁-C₂₀) alkyls, more preferably (C₁-C₁₀)alkyls and most preferably (C₁-C₅) alkyls.

[0045] As defined herein, substituted alkyls are alkyl radicals whereinone or more hydrogen atoms are each independently replaced with anothersubstituent. Typical substituents include, but are not limited to, —R,—OR, —SR, —NRR, —CN, —NO₂, —C(O)R, —C(O)OR, —C(O)NRR, —C(NRR)═NR,—C(O)NROR, —C(NRR)═NOR, —NR—C(O)R, -tetrazol-5-yl, —NR—SO₂—R,—NR—C(O)—NRR, —NR—C(O)—OR, -halogen and -trihalomethyl where each R isindependently —H, (C₁-C₂₀) alkyl, (C₂-C₂₀) alkenyl, (C₂-C₂₀) alkynyl,(C₅-C₂₀) aryl, and (C₆-C₂₆) alkaryl as defined herein.

[0046] As defined herein, alkenyls are unsaturated branched, straightchain or cyclic hydrocarbon radical having at least one carbon-carbondouble bond. The radical may be in a cis or trans conformation about thedouble bond(s). Typical alkenyl groups include, but are not limited to,ethenyl, vinylidene, propenyl, propylidene, isopropenyl, isopropylidene,butenyl, butenylidene, isobutenyl, tert-butenyl, cyclobutenyl, pentenyl,isopentenyl, cyclopentenyl, hexenyl, cyclohexenyl and the like. In apreferred embodiment, the alkenyls of the present invention are (C₂-C₂₀)alkenyls, more preferably (C₂-C₁₀) alkenyls and most preferably (C₂-C₅)alkenyls.

[0047] As defined herein, substituted alkenyls are alkenyl radicalswherein one or more hydrogen atoms are each independently replaced withanother substituent. Typical substituents include, but are not limitedto, —R, —OR, —SR, —NRR, —CN, —NO₂, —C(O)R, —C(O)OR, —C(O)NRR,—C(NRR)═NR, —C(O)NROR, —C(NRR)═NOR, —NR—C(O)R, -tetrazol-5-yl,—NR—SO₂—R, —NR—C(O)—NRR, —NR—C(O)—OR, -halogen and -trihalomethyl whereeach R is independently —H, (₁l-C₈) alkyl, (C₂-C₈) alkenyl, (C₂-C₈)alkynyl, (C₅-C₂₀) aryl, and (C₆-C₂₆) alkaryl as defined herein.

[0048] As defined herein, cycloalkyls are cyclic or polycyclic saturatedor unsaturated hydrocarbon radicals. Typical cycloalkyl groups include,but are not limited to, cyclopropanyl, cyclobutanyl, cyclopentanyl,cyclohexanyl and higher cycloalkyls, adamantyl, cubanyl, prismanyl andhigher polycylicalkyls, and the like. In a preferred embodiment, thecycloalkyls of the present invention are (C₃-C₂₀) cycloalkyls.

[0049] As defined herein, substituted cycloalkyls are cycloalkylradicals wherein one or more hydrogen atoms are each independentlyreplaced with another substituent. Typical substituents include, but arenot limited to, —R, —OR, —SR, —NRR, —CN, —NO₂, —C(O)R, —C(O)OR,—C(O)NRR, —(NRR)═NR, —C(O)NROR, —C(NRR)═NOR, —NR—C(O)R, -tetrazol-5-yl,—NR—SO₂—R, —NR—C(O)—NRR, —NR—C(O)—OR, -halogen and -trihalomethyl whereeach R is independently —H, (C₁-C₈) alkyl, (C₂-C₈) alkenyl, (C₂-C₈)alkynyl, (C₅-C₂₀) aryl, and (C₆-C₂₆) alkaryl as defined herein.

[0050] As defined herein, heterocycloalkyls are cycloalkyl moietieswherein one of the ring carbon atoms is replaced with another atom suchas N, P, O, S, As, Ge, Se, Si, Te, etc. Typical heterocycloalkylsinclude, but are not limited to, imidazolidyl, piperazyl, piperidyl,pyrazolidyl, pyrrolidyl, quinuclidyl, etc. In a preferred embodiment,the cycloheteroalkyl has between 5 and 10 members. Particularlypreferred cycloheteroalkyls are morpholino, tetrahydrofuryl, andpyrrolidyl.

[0051] As defined herein, substituted heterocycloalkyls arecycloheteroalkyl radicals wherein one or more hydrogen atoms are eachindependently replaced with another substituent. Typical substituentsinclude, but are not limited to, —R, —OR, —SR, —NRR, —CN, —NO₂, —C(O)R,—C(O)OR, —C(O)NRR, —C(NRR)═NR, —C(O)NROR, —C(NRR)═NOR, —NR—C(O)R,-tetrazol-5-yl, —NR—SO₂—R, —NR—C(O)—NRR, —NR—C(O)—OR, -halogen and-trihalomethyl where each R is independently —H, (C₁-C₂₀) alkyl,(C₂-C₂₀) alkenyl, (C₂-C₂₀) alkynyl, (C5-C₂₀) aryl, (C₆-C₂₆) alkaryl,5-20 membered heteroaryl, and 6-26 membered alk-heteroaryl as definedherein.

[0052] As defined herein, aryls are unsaturated cyclic hydrocarbonradicals having a conjugated π electron system. Typical aryl groupsinclude, but are not limited to, penta-2,4-dienyl, phenyl, naphthyl,aceanthrylyl, acenaphthyl, anthracyl, azulenyl, chrysenyl, indacenyl,indanyl, ovalenyl, perylenyl, phenanthrenyl, phenalenyl, picenyl,pyrenyl, pyranthrenyl, rubicenyl and the like. In a preferredembodiment, the aryl group is (C₅-C₂₀) aryl, more preferably (C₅-C₁₀)aryl and most preferably phenyl.

[0053] As defined herein, alkaryls are straight-chain (C₁-C₂₀) alkyl,(C₂-C₂₀) alkenyl or (C₂-C₂₀) alkynyl groups wherein one of the hydrogenatoms bonded to the terminal carbon is replaced with an (C₅-C₂₀) arylmoiety. Alkaryls also refer to a branched-chain alkyl, alkenyl oralkynyl groups wherein one of the hydrogen atoms bonded to a terminalcarbon is replaced with an aryl moiety. Typical alkaryl groups include,but are not limited to, benzyl, benzylidene, benzylidyne, benzenobenzyl,naphthalenobenzyl and the like. In a preferred embodiment, the alkarylgroup is (C₆-C₂₆) alkaryl, i.e., the alkyl, alkenyl or alkynyl moiety ofthe alkaryl group is (C₁-C₂₀) and the aryl moiety is (C₁-C₂₀). Inparticularly preferred embodiments, the alkaryl group is (C₆-C₁₃), i.e.,the alkyl, alkenyl or alkynyl moiety of the alkaryl group is (C₁-C₃) andthe aryl moiety is (C.-C₁₀).

[0054] As defined herein, heteroaryls are aryl moieties wherein one ormore carbon atoms have been replaced with another atom, such as N, P, O,S, As, Ge, Se, Si, Te, etc. Typical heteroaryl groups include, but arenot limited to, acridarsine, acridine, arsanthridine, arsindole,arsindoline, benzodioxole, benzothiadiazole, carbazole, β-carboline,chromane, chromene, cinnoline, furan, imidazole, indazole, indole,isoindole, indolizine, isoarsindole, isoarsinoline, isobenzofuran,isochromane, isochromene, isoindole, isophosphoindole, isophosphinoline,isoquinoline, isothiazole, isoxazole, naphthyridine, perimidine,phenanthridine, phenanthroline, phenazine, phosphoindole, phosphinoline,phthalazine, piazthiole, pteridine, purine, pyran, pyrazine, pyrazole,pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline,quinoline, quinolizine, quinoxaline, selenophene, tellurophene,thiazopyrrolizine, thiophene and xanthene.

[0055] As defined herein, alk-heteroaryls are straight-chain alkyl,alkenyl or alkynyl groups where one of the hydrogen atoms bonded to aterminal carbon atom is replaced with a heteroaryl moiety. In apreferred embodiment, the alk-heteroaryl group is a 6-26 memberedalk-heteroaryl, i.e., the alkyl, alkenyl or alkynyl moiety of thealk-heteroaryl is (C₁-C₆) and the heteroaryl moiety is a 5-20-memberedheteroaryl. In particularly preferred embodiments, the alk-heteroarylhas between 6 and 13 members, i.e., the alkyl, alkenyl or alkynyl moietyis (C₁-C₃) and the heteroaryl moiety is a 5-10 membered heteroaryl.

[0056] Preferred organic ligands of the present invention are alkynes,such as acetylene and its derivatives, acetates, acetylacetonates,benzoates, ethylenebis(dithiocarbamates), butadiene, butylates,carboxylates (including formates, butanoates, propionates, pentanoates,hexanoates, octanoates, dodecanoates and decanoates), citrates,cyanoalkyls, alkylhalides, dimethylglyoximes, gluconates, glycinates,lactates, alkyl groups (including methyl, ethyl, propyl, iso-propyl,butyl, t-butyl), alkoxides (including, methoxide, ethoxide, oleates,oxalates, palmitates, phenoxides, phenolsulfonates, p-phenolsulfonates,propylene-bis(dithiocarbamate), salicylates, stearates, tartrates,alkylamines, alkenes (including ethylene, propene, butene), benzene andsubstituted benzenes, cyclobutadiene, cyclopentadiene, pyridine,cycloheptatriene, cyclo-octatetraene and the allyl group.

5.1.3.3 Adducts

[0057] Certain groups may act as counter-ions to the transition metalsand their complexes or form adducts with them in order to form thetransition metal enhancers according to the methods of the presentinvention. Such moieties include, but are not limited to,acetoarsenites, antimonides, arsenates, arsenides, arsenites, borates,carbonates, chromates, chromites, cyanides, cyanates, isocyanates,peroxides, hexafluorosulphates, hydrophosphites, hypophosphites,hydrosulfites, fluoroborates, ferrocyanides, meta-arsenites,metaborates, metaphosphates, nitrates, nitrate hexahydrates, nitrides,nitrites, ortho-arsenates, perchlorates, perchlorate hexahydrates,permanganates, phosphates, phosphides, phosphites, pyrophosphates,selenates, selenides, silicates, stannates, sulfates, sulfides,sulfites, thiocyanates, titanates, tungstates, and composite saltscomprising one or more of the above.

5.1.4 Illustrative Transition Metal Enhancers

[0058] The transition metal enhancers that may be used according to themethods of the present invention include, but are not limited to,cobaltous nitrate, cobaltous oxide, cobaltic oxide, cobalt nitrite,cobaltic phosphate, cobaltous chloride, cobaltic chloride, cobaltouscarbonate, chromous acetate, chromic acetate, chromic bromide, chromouschloride, chromic fluoride, chromous oxide, chromium dioxide, chromicoxide, chromic sulfite, chromous sulfate heptahydrate, chromic sulfate,chromic formate, chromic hexanoate, chromium oxychloride, chromicphosphite, cuprous oxide, cupric oxide, cupric chloride, cuprousacetate, cuprous oxide, cuprous chloride, cupric acetate, cupricbromide, cupric chloride, cupric iodide, cupric oxide, cupric sulfateand cupric sulfide, cupric propionate, cupric acetate, cupricmetaborate, cupric benzoate, cupric formate, cupric dodecanoate, cupricnitrite; cupric oxychloride, cupric palmitate, cupric salicylate,manganese iodide, mangnese sulfate, manganous acetate, manganousbenzoate, manganous carbonate, manganese chloride, manganese bromide,manganese dichloride, manganese trichloride, manganous citrate,manganous formate, manganous nitrate, manganous oxalate, manganesemonooxide, manganese dioxide, manganese trioxide, manganese heptoxide,manganic phosphate, manganous pyrophosphate, manganic metaphosphate,manganous hypophosphite, manganous valerate, ferrous acetate, ferricbenzoate, ferrous bromide, ferrous carbonate, ferric formate, ferrouslactate, ferrous nitrate, ferrous oxide, ferric oxide, ferric acetate,ferric hypophosphite, ferric sulfate, ferrous sulfite, ferrichydrosulfite, ferrous bromide, ferric bromide, ferrous chloride, ferricchloride, ferrous iodide, ferric iodide, nickel acetylacetonate, nickelbromide, nickel carbonate, nickel chloride, nickel cyanide, nickeldibromide, nickel dichloride, nickel dioleate, nickel fluoride, nickelfluoroborate, nickel hydroxide, nickel methylate, nickel nitrate, nickelnitrate hexahydrate, nickel oxide, nickel stearate, nickel sulfate,nickel sulfite, nickel thallate, or nickel salts of other organic acidssuch as ricinoleic acid, cobalt chloride, cobalt fluoride, cobaltnitrate, cobalt sulfate, cobalt octoate, cobalt fluoroborate, cobaltstearate, cobalt oxide, cobalt hydroxide, cobaltous bromide, cobaltouschloride, cobalt butylate, cobaltous nitrate hexahydrate, zinc chloride,zinc acetate, zinc bromide, zinc carbonate, zinc citrate, zinc fluoride,zinc hydroxide, zinc iodide, zinc nitrate, zinc oxide, zinc sulfate, ormixtures thereof. Many other transition metal enhancers are within thescope of the present invention. This section merely serves to illustratesome of the possible transition metal enhancers that are suitable to themethods according to the present invention.

[0059] In a preferred embodiment, the transition metal enhancers of thepresent invention are free metals, complexes, adducts, clusters, and/orsalts of zinc, copper, nickel, cobalt, aluminum or gallium.

[0060] Transition metal enhancers that are especially preferred includezinc and copper containing compounds. More preferably, the transitionmetal enhancer is a zinc, nickel, cobalt, copper, aluminum or galliumhalide. In yet an even more preferred embodiment, the transition metalenhancer is ZnCl₂, NiCl₂, CoCl₂, CuCl₂, AlCl₂, or GaCl₂. Even morepreferably the transition metal enhancer is zinc acetate, zinc chloride,or zinc sulfate.

[0061] In other embodiments, the transition metal enhancer is a zincammonium complex together with its counter ion, zinc antimonide, zincarsenate, zinc arsenide, zinc arsenite, zinc benzoate, zinc borate(Zn₂B₆O₁₁), zinc perborate, zinc bromide, zinc butyrate, zinc carbonate,zinc chromate, zinc chrome, zinc chromite, zinc citrate, zinc decanoate,zinc dichromate, zinc dimer, zinc ethylenebis(dithiocarbamate), zincfluoride, zinc formate, zinc gluconate, zinc glycerate, zinc glycolate,zinc hydroxide, zinc iodide, zinc lactate, zinc methoxyethoxide, zincnaphthenate, zinc nitrate, zinc nitrate hexahydrate, zinc nitratetrihydrate, zinc octanoate, zinc oleate, zinc oxide, zinc pentanoate,zinc perchlorate hexahydrate, zinc peroxide, zinc phenolsulfonate, zincpropionate, zinc propylenebis(dithiocarbamate), zinc stannate, zincstearate, zinc sulfate, zinc titanate, zinc tetrafluoroborate, zinctrifluoromethanesulfonate, and enhancers thereof.

[0062] The delivery of a nucleic acid and a transition metal enhancer iscarried out using techniques known in the art of biotechnology asdescribed below.

5.2 Buffers Useful For Nucleic Acid Delivery

[0063] The optimal pH range for a nucleic acid/transition metal enhancermixture may vary depending upon the composition of the nucleic acid, thetype of transition metal enhancer, and the particular cell typereceiving the mixture.

[0064] In one embodiment, the nucleic acid/transition metal enhancersolution is not buffered. In other embodiments, however, the solutionmay be buffered. One or more buffers may be used, for example, toprovide stable conditions for storage of the nucleic acid/transitionmetal enhancer mixture for an extended duration. Any buffer or pH notsubjecting the nucleic acid to any condition of degradation may be usedin the methods of the present invention. If nonnaturally occurringnucleic acids are used, the desirable buffer may be one that issubstantially different than those used in conventional gene therapy.Representative buffers that could be used to buffer the nucleicacid/transition metal enhancer mixture of the present invention include,but are not limited to, N-[carbamoylmethyl]-2-aminoethanesulfonic acid(ACES), N-2[2-acetamido]-2-iminodiacetic acid (ADA),2-amino-2-methyl-2,3-propanediol, 2-amino-2-methyl-1-propanol,3-amino-1-propanesulfonic acid, 2-amino-2-methyl-1propanol,3-[(1,1-dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid(AMSO), N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid (BES),N,N-bis[2-hydroxyethyl]glycine (BICINE),bis[2-hydroxyethyl]iminotris-[hydroxymethyl]methane (BIS-TRIS);1,3-bis[tris(hydroxymethyl)-methylamino]propane (BIS-TRIS PROPANE),4-[cyclohexylamino]-1-butanesulfonic acid (CABS),3-[cyclohexylamino]-1-propanesulfonic acid (CAPS),3-[cyclohexylamino]-2-hydroxy-1-propanesulfonic acid (CAPSO),2-[N-cyclohexylamino]ethanesulfonic acid (CHES),3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO),N-[2-hydroxy-ethyl]-piperazine-N′-[3-propanesulfonic acid] (HEPPS),N-[2-hydroxyethyl]piperazine-N′-[4-butanesulfonic acid] (HEPBS),N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid (HEPES),N-[2-hydroxyethyl]piperazine-N′-[2-hydroxypropanesulfonic acid](HEPPSO), imidazole, 2-[N-morpholino]ethanesulfonic acid (MES),4-[N-morpholino]butanesulfonic acid (MOBS),3-[N-morpholino]propanesulfonic acid (MOPS),3-[N-morpholino]-2-hydroxypropanesulfonic acid (MOPSO),piperazine-N,N′-bis[2-ethanesulfonic acid] (PIPES),piperazine-N,N′-bis[2-hydroxypropanesulfonic acid (POPSO),N-tris[hydroxy-methyl]methyl-4-aminobutanesulfonic acid (TABS),N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS),3-[N-tris(hydroxymethyl)methylamino]-2-hydroxy-propanesulfonic acid(TAPSO), triethanolamine (TEA),N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid (TES),N-tris[hydroxymethyl]methylglycine (TRICINE), triethanolamine,tris[hydroxymethyl]aminomethane (TRIZMA) phosphate, acetate, citrate,borate, and bicarbonate.

[0065] Furthermore, the buffers may be in the form of the free acid,base or salt. For example, if the buffer used occurs as an acid, thebuffer may be, for example, in the form of the acid, sodium salt,disodium salt, hemisodium salt, sodium salt hydrate, potassium salt,dipotassium salt, sesquisodium salt, or any other salt. If the bufferoccurs as a base, the buffer may be, for example, in the form of thefree base or as the hydrochloride. Other buffers may be used to bufferthe nucleic acid/transition metal enhancer solution and the buffersprovided herein merely serve to illustrate representative embodiments ofthe present invention.

[0066] In a preferred embodiment of the presently disclosed method, thenucleic acid/transition metal enhancer mixture is not buffered and thepH is not regulated. In a more preferred embodiment, the pH of thebuffer is between about 4.0 and about 9.0. Even more preferably, the pHis between about 5.5 and 8.5.

5.3 Ratio of Transition Metal Enhancer to Nucleic Acid in the NucleicAcid/Transition Metal Enhancer 5.3.1 Ratio of Nucleic Acid to TransitionMetal Enhancer and Preferred Concentrations of Transition Metal Enhancer

[0067] The ratio of transition metal enhancer to nucleic acid in thenucleic acid/transition metal enhancer mixtures of the present inventioncan vary over a tremendous range because of the large range in nucleicacid size that may be used in the present invention. Thus, depending onthe size of the nucleic acid introduced, the ratio of transition metalenhancer to nucleic acid may be about one mole of transition metalenhancer per ten thousand moles of nucleic acid in the mixture to aboutone mole of transition metal enhancer per 0.0001 moles of nucleic acidin the formulation. Alternatively, the amount of transition metalenhancer relative to nucleic acid in the formulation may be calculatedrelative to the number of base pairs present in the formulation. In suchinstances, the amount of transition metal enhancer in the formulationmay range from about one mole of transition metal enhancer for every tenthousand moles of base pairs in the formulation to about one mole oftransition metal enhancer for every 0.0001 moles of nucleic acid in theformulation.

[0068] In a preferred method of computation, the amount of transitionmetal enhancer and the amount of nucleic acid present in the nucleicacid/transition metal enhancer mixture are considered independent. Theconcentration of the transition metal enhancer in the nucleicacid/transition metal enhancer mixture may range from about 0.01 mM to250 mM if the mixture is in liquid form. More preferably, theconcentration of the transition metal enhancer in the mixture is about0.1 mM to about 6.0 mM. If the mixture is a lyophilized powder, theconcentration of transition metal enhancer in the reconstituted mixtureis about 0.01 mM to 250 mM. More preferably, the concentration of thetransition metal enhancer in the reconstituted mixture is 0.1 mM toabout 6.0 mM.

[0069] Some of the transition metals of the present invention, such aszinc, are essential trace elements that are present in most life forms.Therefore, it is expected that some of the transition metal enhancers ofthe present invention may be found in most bodily fluids and other invivo environments. However, the concentrations of transition metalenhancer used in the present invention are considerably higher than theconcentrations of transition metal enhancers that are found in a naturalin vivo environment. For example, although dependent on a person's diet,the amount of zinc in human blood is about 880 μg/100 mL or about 0.135mM. See, e.g., Altman et al., Blood and Other Body Fluids, Federation ofAmerican Societies for Experimental Biology. Such a concentration isconsiderably less than the concentrations of transition metal enhancerrequired in the nucleic acid/transition metal enhancer mixtures of thepresent invention.

5.3.2 Amount of Nucleic Acid Administered

[0070] The amount of nucleic acid applied according to the methods ofthe present invention will vary greatly according to a number of factorsincluding, but not limited to, the susceptibility of the target cells tonucleic acid uptake, the levels of protein expression desired, if any,and the clinical status requiring the gene therapy. For example, theamount of nucleic acid injected into a salivary gland of a human isgenerally from about 1 μg to 200 mg, preferably from about 100 μg to 100mg, more preferably from about 500 μg to 50 mg, most preferably about 20mg. The amount of nucleic acid injected into the pancreas of a human maybe, for example, from about 1 μg to 750 mg, preferably from about 500 μgto 500 mg, more preferably from about 10 mg to 200 mg, most preferablyabout 40 mg. The amounts of nucleic acid suitable for human gene therapymay be extrapolated from the amounts of nucleic acid effective for genetherapy in an animal model. For example, the amount of nucleic acid forgene therapy in a human is known to be about one to two hundred timesthe amount of nucleic acid effective in gene therapy in a rat.Furthermore, the amount of nucleic acid necessary to accomplish celltransfection will decrease with a corresponding increase in theefficiency of the transfection method used. In one preferred embodiment,the total concentration of the nucleic acid in the final mixture is fromabout 0.1 μg/ml to about 15 mg/ml.

5.3.3 Amount of Cationic Lipid Administered

[0071] In some embodiments of the present invention, the nucleic acid ismixed with cationic lipids and transition metal to form a cationiclipid/DNA/transition metal mixture. The cationic lipid/DNA/transitionmetal mixture is then used to transfect target cells of interest. Inparticular, it has been found that the use of cationic lipids incombination with a transition metal is particularly useful for the invitro transfection of nucleic acids.

[0072] In embodiments in which a nucleic acid is delivered to targetcells in the form of a cationic lipid/DNA/transition metal mixture,preferred nucleic acid concentrations range from 0.1 to 25 μg/μL. Morepreferably, the nucleic acid concentration is from 0.5 to 10 μg/μL.Furthermore, the transition metal concentration is preferably from about0.01 to about 10 mM. However, the precise range of useful transitionmetal concentration range is largely determined by the uniquecharacteristics specific transition metal used, such as solubility.

[0073] The present invention encompasses liposome solutions containing(i) a single form of cationic lipid, (ii) lipid mixtures that include acationic lipid component and a neutral lipid component, or (iii)mixtures of different cationic lipids. An example of a lipid mixturethat includes a cationic lipid component and a neutral lipid componentis a mixture of dioleoylphosphatidyl ethanolamine and cholesterol. Asused herein, the term cationic lipid is broadly construed and includes,but is not limited to, any lipid that contains functionality, such asprimary amine, secondary amine, tertiary amine, or quaternary ammoniumgroup, having a net positive charge at a useful physiological pH.Examples of cationic lipids include 1:1N,N-[Bis(2-hydroxyethyl)]-N-methyl-N-[2,3-bis(tetradecanoyloxy)propyl]ammoniumchloride andN,N,N′,N′-tetramethyl-N,N′-bis(2-hydroxyethyl)-2,3-bis(9(z)-octadecenoyloxy)-1,4-butanediaminiumiodide. Additional cationic lipids can be found in references such asThe Lipid Handbook, Gunstone, Harwood, and Padley (Eds.), SecondEdition, July 1994, CRC Press.

[0074] In some embodiments of the present invention, the amount ofliposome present in cationic lipid/DNA/transition metal mixture isexpressed in terms of the cationic lipid:DNA phosphate charge ratio.Illustrative complexes include those having a charge ratio of 0.5, 0.75,1.0, and 2.0. Preferably, the cationic lipid:DNA phosphate charge ratioranges from 0.01 to 12. More preferably, the lipid:DNA phosphate chargeratio ranges from 0.1 to 6. Even more preferably, the lipid:DNAphosphate charge ratio ranges from 0.5 to 4.

[0075] An important advantage of the present invention over prior artsystems is that liposomes having low lipid:DNA phosphate charge ratios(i.e. less than 1) are still efficacious in delivering nucleic acids tocells.

5.4 Nucleic Acids That Can Be Delivered

[0076] Nucleic acids that may be used to form the nucleicacid/transition metal enhancers described in the present inventioninclude DNA, DNA vectors, RNA, and synthetic oligonucleotides. All ofthese nucleic acids may either occur naturally or may be constructed ormodified by the techniques known in the art of molecular biology andchemistry. The nucleic acids may exist as a circular or linear form, oralternatively, may be branched. The nucleic acid may be single stranded,double stranded, or may form other, more complex structures. The nucleicacid may carry a positive, neutral, or negative charge, although it willmost preferably have a negative charge. In a preferred embodiment, thereis no limit on the size range of the nucleic acids. In an even morepreferred embodiment the nucleic acid will be from about 10 to about20,000 nucleotides in length. In one preferred embodiment the nucleicacid will be from about 100 to about 10,000 nucleotides. In an even morepreferred embodiment, the nucleic acid will comprise from about 500 toabout 5,000 nucleotides.

5.4.1 Use of DNA Vectors as the Source of Nucleic Acid

[0077] The DNA vectors that can be used to form the nucleicacid/transition metal enhancer mixtures according to the presentinvention will typically be constructed from heterologous DNA sourcesusing standard recombinant DNA techniques well known in the art. Variousknown vectors, such as DNA viral vectors, bacterial vectors, and vectorscapable of replication in both eukaryotic and prokaryotic hosts, can beused in accordance with the present invention. Depending on the desiredresult, the vectors may contain sequences that mediate the stableintegration of the vector DNA into a specific site in a particularchromosome. Such integration may provide the possibility for long-term,stable expression of genes contained within the vectors and/or enable achange in the genome that is beneficial. Alternatively, the vectors maybe designed so that they do not insert into the cellular genome. Vectorsthat do not insert into the genome may or may not contain sequences toallow them to replicate within the cell. Thus, by varying the componentsincluded within the sequence of the DNA vectors, the stability and copynumber of the vectors in the cells can be controlled as desired.

[0078] The vectors useful for the present invention will typicallycontain one or more genes or gene fragments of interest to allow theexpression of one or more gene products following transfer of the vectorinto a target cell. In addition to these genes, vectors may also containone or more marker genes to allow for selection, under specific growthconditions, of cells containing the vector DNA or to allow cellscarrying vector sequences to be identified. Expression of an introducedgene or gene fragment can be controlled in a variety of ways, dependingon the desired result and the construction of the vector. The gene maybe expressed constitutively at various levels in the cells, or it may beexpressed only under specific physiologic conditions or in specific celltypes. Expression depends on the presence of a promoter region upstreamfrom the gene, and may also be controlled by enhancer regions and otherregulatory elements within the vector or within adjacent regions of thegenomic DNA. The construction of DNA vectors for gene therapy and thecomponents necessary for replication of the vectors, for insertion ofthe vectors into the cell genome, and for expression of genes carried bythe vectors is well known in the art. See Curiel et al., Am. J. Respir.Cell Mol. Biol. 14:1, 1996; German et al., U.S. Pat. No. 5,837,693.

[0079] The primary expression product from a gene carried by a DNAvector is RNA. If the targeted cells are deficient in a particulartransfer RNA or ribosomal RNA, the vector may complement this defectdirectly by providing a gene encoding the desired transfer or ribosomalRNA. Most typically, however, the RNA expressed from the gene carried bythe vector DNA will function as a messenger RNA and encode a protein orprotein fragment. Depending on the targeting sequences contained withinthe primary structure of the protein, the expressed protein will eitherbe secreted from the cell, will be transported to one of theintracellular organelles, or will remain in the cytosol. Amino acidsequences within the expressed protein may also direct othermodifications to the protein during or after translation of the protein.Proteins expressed from vector DNA may provide a therapeutic effect tothe targeted cell or to other cells in the organism.

[0080] Depending on the sequence and stability of an RNA produced from agene carried by the DNA vector, the RNA may also have antisense activitywithin the cell. Antisense oligonucleotides are typically designed tobind specifically to mRNA molecules within the cell to increase ordecrease the stability or translation efficiency of the bound mRNA. Itwill be appreciated by those of skill in the art, however, that otherforms of nucleic acid, including other RNA molecules and genomic DNA,may also be targeted by antisense methods. The target sequence to whichan antisense RNA is complementary may be derived from a virus or apathogenic microorganism, and expression of the antisense RNA encoded bya DNA vector and delivered into a target cell by methods of theinvention may provide protection and/or a cure from infection caused bythe virus or pathogenic microorganism. Alternatively, the targetsequence to which the antisense RNA is complementary may be encoded bythe target cell itself, and expression of the antisense RNA may protectan organism from disease states caused by abnormal expression of atargeted gene in the targeted cell. Target genes may include the variousoncogenes and proto-oncogenes, as well as genes coding for theamyloid-like protein associated with Alzheimer's disease, the prionprotein, and others. See Padmapriya et al., U.S. Pat. No. 5,929,226.

[0081] The RNA produced from the gene carried by the DNA vector may alsofunction as a ribozyme. Ribozymes are RNA molecules that catalyze thehydrolysis of phosphodiester bonds in other RNA molecules. They canthereby inhibit and/or reduce the activity of a target RNA to which theybind. Ribozymes offer two significant advantages over antisense RNAmolecules in gene therapy. First, because the activity of a ribozyme iscatalytic, and a single ribozyme molecule can, therefore, cleave manytarget RNAs, ribozymes may be more efficient than antisense RNAs andmay, therefore, be effective at lower concentrations. Second, becausesingle mismatches can disrupt the catalytic activity of a ribozyme butwould not necessary disrupt the binding of an antisense RNA tonon-target RNA, the specificity of action of a ribozyme is greater thanthat of an antisense RNA. See Chowrira et al., U.S. Pat. No. 5,837,855.

5.4.2 Use of RNA as the Source of Nucleic Acid

[0082] Although it is possible to express an RNA of interest as derivedfrom genes carried by DNA vectors, it may be desirable for purposes ofthe invention to use RNA itself to form the nucleic acid/transitionmetal enhancer mixtures. Large quantities of RNA can typically begenerated by transcription from linear DNA templates using various RNApolymerases in a cell-free system. The DNA templates are constructed toencode the desired RNA sequences using techniques known in the art ofmolecular biology. The gene to be expressed is generally flanked by anRNA polymerase-specific promoter on its 5′end and a template encoding apolyA tail and transcription termination sequences on its 3′end. Thegene is transcribed by RNA polymerase in the presence of a 5′cap and thefour nucleoside triphosphates. It may be desirable to purify the RNAfollowing its transcription to remove the polymerase and unincorporatedsmall molecules. In addition, various chemical and enzymatic methods canbe used to modify the RNA molecules included in the nucleicacid/transition metal enhancer solutions in order to protect them fromnuclease digestion and to increase their stabilities within cells.Possible methods include end modification and circularization. SeeFelgner et al, U.S. Pat. No. 5,703,055.

[0083] RNA generated in any manner can be used to produce nucleicacid/transition metal enhancer mixtures and be delivered to target cellsas provided by the methods of the invention. Transfer and ribosomal RNAscan be delivered into cells lacking sufficient quantities of thesemolecules. Likewise, messenger RNAs can be delivered into cells to allowexpression of their encoded proteins. In addition, antisense RNAmolecules and ribozymes produced by RNA polymerase can be delivered totarget cells to provide any desired therapeutic effect.

[0084] RNA in the form of retroviral vectors or modified retroviralvectors can also be used to form the nucleic acid/transition metalenhancer mixtures of the invention. Retroviruses carry their geneticinformation in the form of RNA and can be used to express genes or genefragments of interest in eukaryotic cells. Upon entering a cell, theretroviral RNA is reverse transcribed into DNA, and the DNA issubsequently inserted into the genomic DNA of the infected cell. Genesor gene fragments carried by a retrovirus and placed under the controlof an appropriate promoter can, therefore, be expressed in cells asdescribed above for DNA vectors.

5.4.3 Use of Synthetic Oligonucleotides and Analogues as a Source ofNucleic Acid

[0085] The methods according to the present invention may also beperformed using synthetic oligonucleotides and /or analogues to generatenucleic acid/transition metal enhancer mixtures. In particular,oligonucleotides synthesized by standard solid-phase chemical methodsmay be used. These molecules may additionally contain non-naturalnucleic acid base analogues, sugar analogues, or linkages, or they maybe modified by chemical means prior to formation of the mixtures. Thesealterations may result in an improvement in one or more desiredproperties for the oligonucleotides, such as an improved delivery of theoligonucleotides into target cells or an increased stability of theoligonucleotides within the cells. See Padmapriya et al., U.S. Pat. No.5,929,226.

[0086] The heterocyclic bases of the oligonucleotide may include thenaturally occurring bases (adenine, cytosine, guanine, thymine, anduracil) or may include synthetic modifications or analogues of thesebases. The sugar component of the oligonucleotide may include thenaturally occurring sugars (ribose and 2′-deoxyribose) or may includesynthetic modifications or analogues of these sugars. In addition, theanomeric configuration of the sugar and even the position of coupling ofthe base to the sugar can be natural or non-natural in theoligonucleotides used to make the nucleic acid/transition metal enhancermixtures of the invention. Finally, the linkage between nucleosides,modified nucleosides, or nucleoside analogues within the oligonucleotidemay include the naturally occurring linkage (5′ to 3′ phosphodiester) ormay include synthetic modifications or analogues of this linkage. Thoseskilled in the art will recognize that a large number of nucleosides,modified nucleosides, and nucleoside analogues are known in the priorart, and that any of these can be used alone or in combination togenerate oligonucleotides for use in the nucleic acid/transition metalenhancer mixtures contemplated in the invention.

[0087] The design and synthesis of an oligonucleotide will likewise varydepending on the desired effects of the oligonucleotide within the cell.As described above for antisense RNA, antisense oligonucleotides can bedesigned to bind specifically to a target mRNA, and the binding mayincrease or decrease the stability or translation efficiency of thebound mRNA. Depending on the target, the method can potentially be usedto control infection by a virus or pathogenic microorganism or can beused to regulate the growth of cells having desirable or undesirableproperties. Alternatively, an oligonucleotide may be designed torecognize and bind to double-stranded DNA, and the triple helix formedas a consequence of this binding may alter expression of a gene targetedby the method. Finally, oligonucleotides delivered into a cell by themethods of the invention may have new functions, such as a novelcatalytic activity or binding ability, and should not be limited tothose functions known in the prior art.

5.5 Administration of the Nucleic Acid/Transition Metal Enhancer Mixture

[0088] In some embodiments of the present invention, such as embodimentsdirected to in vivo gene therapy methods, the nucleic acid/transitionmetal enhancer mixture may be directly administered to the cells withinthe organism of interest. In this instance, the dosage to beadministered varies with the condition and size of the subject beingtreated as well as the frequency of treatment and the route ofadministration. Desirable regimens for chronic therapy protocols,including suitable dosage and frequency of administration, may be guidedby the subjects initial response to the enhancer in view of soundclinical judgment. In some embodiments, the parenteral route ofinjection into the interstitial space either directly or via thebloodstream is used, although other parenteral routes, such asinhalation of an aerosol formulation, may be required in theadministration to specific cells, as for example to the mucous membranesof the nose, throat, bronchial tissues or lungs.

[0089] In a preferred embodiment, a formulation comprising the nucleicacid and transition metal enhancer in a aqueous carrier is injected invivo into the tissue in amounts of from about 10 μl per site to about100 ml per site.

5.6 Representative Nucleic Acid Delivery Methods to Various Tissues5.6.1 Nucleic Acid Delivery to Secretory Glands

[0090] The methods of the present invention may be used to delivernucleic acids to various secretory glands using routes of administrationsuch as, for example, those described by German et al., U.S. Pat. No.5,885,971. As used herein, a secretory gland is defined as aggregationof cells specialized to secrete or release materials not related totheir ordinary metabolic needs. Secretory glands may include salivaryglands, pancreas, mammary glands, thyroid gland, thymus gland, pituitarygland, liver, and other glands well known to one skilled in the art.

[0091] In one embodiment, the methods of the present invention are usedto deliver nucleic acids to salivary glands. Salivary glands are definedherein as any gland of the oral cavity that secretes saliva, includingthe glandulae salivariae majores of the oral cavity (collectively, theparotid, sublingual, and submandibular glands) and the glandulacsalivariae minores of the tongue, lips, cheeks, and palate (labial,buccal, molar, palatine, lingual, and anterior lingual glands).

[0092] The routes of administration, described by German et al., for thepresently claimed nucleic acid/transition metal enhancer mixture mayinclude administration according to known in vivo or ex vivo methods.When in vivo methods are used, the nucleic acid/transition metalenhancer mixture may be injected directly into a secretory gland or intoa secretory gland duct. The subsequent exposure of the secretory glandto the nucleic acid/transition metal enhancer mixture results in theuptake of the nucleic acid by the target cells present within theaggregation comprising the secretory gland.

[0093] Alternatively, if ex vivo methods are used to introduce nucleicacid to any of the described secretory glands, a biopsy of secretorygland tissue may be obtained from the organism of interest. In apreferred embodiment, the organism is a mammal. Preferably, the biopsyis used to establish a primary cell culture according to known. Thebiopsy tissue or the primary cell culture then receives the nucleicacid/transition metal enhancer mixture, resulting in the uptake of thenucleic acid in to the internal cellular environment of the secretorygland cells. Cells that have been exposed to the nucleic acid/transitionenhancer metal mixture are then reintroduced into the secretory glandwithin the organism.

[0094] When the nucleic acid contains certain types of retroviralsequences known to those skilled in the art, a portion of the nucleicacid may also be incorporated into the genome of the secretory glandcells. The incorporation of the exogenous nucleic acid into the genomeof the secretory gland cell typically results in the stabletranscription of a portion of nucleic acid that is operably linked to apromoter. In a preferred embodiment, however, the nucleic acid in thenucleic acid/transition metal enhancer mixture does not contain anyretroviral sequences and is only transiently transcribed.

[0095] If the nucleic acid is transcribed by the cell, and codes for apolypeptide, the polypeptide may be then expressed by the cellularmachinery after gene delivery. The polypeptide may be a functionalprotein and may be secreted by the secretory cells into the bloodstream,gastrointestinal system or interstitial spaces or any other internal orexternal compartment of the organism. Therefore, the methods of thepresent invention could be used to supplement various proteins ofinterest in the bloodstream with the host organism by the addition ofthe newly transcribed peptide product. Such an application offersutility in treatment of a wide variety of diseases such as, for example,those described by German et al., U.S. Pat. No. 5,837,693. Accordingly,representative examples of proteins that may be encoded by the nucleicacid in the nucleic acid/transition metal enhancer mixture include, butare not limited to, insulin, human growth hormone, erythropoietin,clotting factor VII, bovine growth hormone, platelet derived growthfactor, clotting factor VIII, thrombopoietin, interleukin-1,interluekin-2, interleukin-1 RA, superoxide dismutase, catalase,fibroblast growth factor, neurite growth factor, granulocyte colonystimulating factor, L-asparaginase, uricase, chymotrypsin,carboxypeptidase, sucrase, calcitonin, Ob gene product, glucagon,interferon, transforming growth factor, ciliary neurite transformingfactor, insulin-like growth factor-1, granulocyte macrophage colonystimulating factor, brain-derived neurite factor, insulintropin, tissueplasminogen activator, urokinase, streptokinase, adenosine deamidase,calcitonin, arginase, phenylalanine ammonia lyase, γ-interferon, pepsin,trypsin, elastase, lactase, intrinsic factor, cholecystokinin, andinsulinotrophic hormone.

5.6.2 Nucleic Acid Delivery to the Brain

[0096] The methods according to the present invention may be used todeliver a nucleic acid to various desired regions of brain tissue usingroutes of administration as described by U.S. Pat. No. 5,580,859 toFelgner et al. and U.S. Pat. No. 5,916,803 to Sedlacek et al. As usedherein, brain tissue is generally defined as an aggregation of cells,including, but not limited to neurons, Schwann cells, glial cells andastrocytes. Such cells are known to contain properties which arespecialized to perform various functions associated with the central orperipheral nervous systems. Preparations to be used according to themethods of presently claimed invention may also be introduced intovarious nerve cells using known approaches described previously.

[0097] In one embodiment, brain tissue may be isolated from adult micefollowing injection of a gene construct comprising a sequence encoding,for example, a polypeptide as described above. In one embodiment, apromoter is operably associated with a sequence encoding a molecule,such as a polynucleotide. More specifically, other molecules which maybe practiced according to the present invention may be polynucleotidesincluding genomic DNA, cDNA, and mRNA that encode therapeutically usefulproteins known in the art, ribosomal RNA, antisense RNA or DNApolynucleotides, that are useful to inactivate transcription products ofgenes, or even retroviral nucleic acid. The injections may beadministered through various administration routes as described hereinto a desired region, for example, into each of the bilateral parietal,frontal, temporal or visual cortex regions. Following injection of thegenetic material, the tissue may be assayed in accordance with themethods disclosed herein. Successful introduction of the geneticmaterial upon analysis of gene expression may provide necessaryinformation to direct therapeutic strategies such as, for example, toinduce, enhance and/or inhibit the formation, growth, proliferation,differentiation, maintenance of neurons and/or related neural cells andtissues such as brain cells, Schwann cells, glial cells and astrocytes.

[0098] As previously described, the nucleic acid may contain any of thedesired retroviral sequences or various exogenous nucleic acid sequencesthat are able to be incorporated into the genome of the nerve cell,thereby resulting in the stable transcription of a portion of nucleicacid. In a preferred embodiment, however, the nucleic acid in thenucleic acid/transition metal enhancer mixture does not contain anyretroviral sequences and is only transiently transcribed. Alternatively,the nucleic acid codes for a polypeptide that may be expressed by thecellular machinery. The polypeptide may be a functional protein and maybe secreted by the nerve cells into the interstitial spaces within thebrain. Therefore, the methods of the present invention may be used tosupplement various proteins present within the host organism by theaddition of the newly transcribed peptide product in a manner asdescribed above.

[0099] Another embodiment of the present invention is a therapeuticmethod and composition for treating disorders of neurons and/or relatedneural cells and tissues associated with Schwann cells, glial cells andastrocytes, and other conditions related to neuronal and neural tissuedisorders or diseases. The invention is further directed to therapeuticmethods for repair and restoration of nerve tissue.

[0100] It is further contemplated that the methods of the presentinvention may increase neuronal, glial cell and astrocyte survival andtherefore have great utility in known transplantation protocols for thetreatment of conditions known to cause a decrease in neuronal survival.

5.6.3 Nucleic Acid Delivery to Muscle

[0101] The methods of the present invention may be used to deliver anucleic acid to various desired regions of muscle tissue using routes ofadministration as described by U.S. Pat. Nos. 5,580,859 and 5,916,803.As used herein, muscle tissue is generally defined as an aggregation ofcells, which comprise the bulk of the body's musculature including, butnot limited to cardiomyocytes, skeletal and smooth muscle cells. Suchcells are known to have properties which are specialized to performvarious functions commonly associated with movement as well as otherknown functions of the muscular system. Preparations to be usedaccording to the methods of the presently claimed invention can also beintroduced into various muscle cells using known approaches describedabove.

[0102] In one embodiment, muscle tissue may be isolated from adult micefollowing injection of a gene construct comprising a sequence encoding,for example, a polypeptide. In one embodiment, a promoter is operablyassociated with a sequence encoding the polypeptide. More specifically,other molecules which may be practiced according to the presentinvention may be similar to those described previously.

[0103] Administration of the nucleic acid/transition metal enhancermixture according to the present invention may be to a desired region,such as a particular muscle group within the organism, or a particularlocation within such a muscle group. Following injection of the geneticmaterial, the muscle tissue may be assayed in accordance with themethods described previously. Successful introduction of the geneticmaterial as demonstrated by measurable gene expression may provideinformation useful for developing therapeutic strategies such as, forexample, to induce, enhance and/or inhibit the formation, growth,proliferation, differentiation, maintenance of the various cells of theskeletalmuscular system, including cardiomyocytes, skeletal and smoothmuscle cells.

[0104] As previously described, the nucleic acid may contain any of thedesired retroviral sequences or various exogenous nucleic acid sequencesthat are able to be incorporated into the genome of the muscle cell,thereby resulting in the stable transcription of a portion of thenucleic acid. In a preferred embodiment, however, the nucleic acid inthe nucleic acid/transition metal enhancer mixture does not contain anyretroviral sequences and is only transiently transcribed. Alternatively,the nucleic acid codes for a polypeptide that may be expressed by thecellular machinery. The polypeptide may be a functional protein and maybe secreted by the muscle cells into the interstitial spaces of thebrain. Therefore, the methods of the present invention may be used tosupplement various proteins present within the host organism by theaddition of the newly transcribed peptide product in a manner asdescribed above.

[0105] Another embodiment of the present invention is a therapeuticmethod for treating disorders of myocytes and/or related muscle cellsand tissues such as cardiomyocytes, skeletal and smooth muscle cells,and any other condition related to a muscular tissue disorder ordisease. The invention is further directed to therapeutic methods forrepair and restoration of muscular tissue.

[0106] It is further contemplated that the methods of the presentinvention may increase muscle cell survival and therefore be useful inknown transplantation procedures and for the treatment of conditionsknown to cause any degeneration in related tissues.

5.6.4 Nucleic Acid Delivery to the Pancreas

[0107] The methods of the present invention may be used to deliver anucleic acid to various desired regions of pancreatic tissue usingroutes of administration as described by U.S. Pat. Nos. 5,580,859 and5,916,803. As used herein, pancreatic tissue is defined as comprising anendocrine portion (the pars endocrina) and an exocrine portion (the parsexocrina). The pars endocrina, contains the islets of Langerhans, andthe pars exocrina contains acinar cells. The pancreas is generallydefined as an aggregation of cells, which comprises the entirepancreatic structure, including but not limited to ductal cells, acinarcells, beta cells, alpha cells, and other cells of the Islets ofLangerhans. Such cells are known to have properties which arespecialized to perform various functions commonly associated withdigestive processes, hormonal regulation, and other known functions.

[0108] Compositions described according to the methods of the presentlyclaimed invention can also be introduced into various pancreatic cellsusing known approaches described above. The routes of administration,described by German et al., for the presently claimed nucleicacid/transition metal enhancer mixture are used in accordance with knownex vivo or in vitro methods.

[0109] In one embodiment, pancreatic tissue may be isolated from adultmice following the successful injection of a gene construct compositionin accordance with the present invention. The genetic construct maycomprise a sequence encoding, for example, a polypeptide. In oneembodiment, a promoter is operably associated with a sequence encodingthe polypeptide. More specifically, other molecules which may bepracticed according to the present invention may be similar to thosedescribed previously.

[0110] Administration of the nucleic acid/transition metal enhancermixture according to the present invention may be to a desired regionwithin the pancreatic structure, such as a specialized cell group withinthe pancreas. Following injection of the genetic material, thepancreatic tissue may be assayed in accordance with the known methodsdesigned to quantitate protein levels or other methods for detectingincreased gene expression. Successful introduction of the geneticmaterial as demonstrated by measurable gene expression may provideinformation useful for developing therapeutic strategies such as, forexample, to induce, enhance and/or inhibit the formation, growth,proliferation, differentiation, maintenance of the various cells of thepancreas, including acinar cells, beta cells, alpha cells, ductal cells,and other cells of the Islets of Langerhans.

[0111] If the nucleic acid is transcribed by the cell, and codes for apolypeptide, the polypeptide may be expressed by the cellular machineryafter gene delivery. The polypeptide may be a functional protein and maybe secreted by pancreatic cells into the bloodstream, gastrointestinalsystem or interstitial spaces or any other internal or externalcompartment of the organism. Therefore, the methods of the presentinvention could be used to supplement various proteins of interest inthe bloodstream with the host organism by the addition of the newlytranscribed peptide product. Such an application offers utility intreatment of a wide variety of diseases such as, for example, thosedescribed by German et al., U.S. Pat. No. 5,837,693. Accordingly,representative examples of proteins that my be encoded by the nucleicacid in the nucleic acid/transition metal enhancer mixture include, butare not limited to, insulin, human growth hormone, erythropoietin,clotting factor VII, bovine growth hormone, platelet derived growthfactor, clotting factor VIII, thrombopoietin, interleukin-1,interleukin-2, interleukin-1 RA, superoxide dismutase, catalase,fibroblast growth factor, neurite growth factor, granulocyte colonystimulating factor, L-asparaginase, uricase, chymotrypsin,carboxypeptidase, sucrase, calcitonin, Ob Gene product, glucagon,interferon, transforming growth factor, ciliary neurite transformingfactor, insulin-like growth factor-1, granulocyte macrophage colonystimulating factor, brain-derived neurite factor, insulintropin, tissueplasminogen activator, urokinase, streptokinase, adenosine deamidase,calcitonin, arginase, phenylalanine ammonia lyase, χ-interferon, pepsin,trypsin, elastase, lactase, intrinsic factor, cholecystorkinin, andinsulinotrophic hormone.

[0112] Another embodiment of the present invention is a therapeuticmethod for treating disorders associated with pancreatic celldegeneration and any other condition related to a pancreatic tissuedisorder or disease. The invention is further directed to therapeuticmethods for repair and restoration of defective pancreatic tissue.

[0113] It is further contemplated that the methods of the presentinvention may increase pancreatic cell survival and therefore be usefulin known transplantation procedures and for the treatment of conditionsknown to cause any degeneration in related tissues.

5.6.5 Nucleic Acid Delivery to Other Tissue Types

[0114] The present invention presents methods using a nucleicacid/transition metal enhancer mixture that facilitates intracellulardelivery of therapeutically effective amounts of nucleic acid to targetcells. The therapeutic enhancer and the method of use in gene deliveryas presently claimed may be further suitable for use with other celltypes including, but not limited to, cell groups associated with thebreast, thyroid, bone, bladder, skin, liver, stomach, lung, kidney,gastrointestinal tract, and various reproductive organs such as thetestes, uterus and ovaries. Successful introduction of the geneticmaterial resulting in subsequent gene expression may provide usefulinformation for developing therapeutic strategies such as, for example,to induce, enhance and/or inhibit the formation, growth, proliferation,differentiation, maintenance of the various cells of the tissuesdescribed above.

[0115] As previously described, the nucleic acid may contain any of thedesired retroviral sequences or various exogenous nucleic acid sequencesthat are able to be incorporated into the genome of the muscle cell,thereby resulting in the stable transcription of a portion of thenucleic acid. In a preferred embodiment, however, the nucleic acid inthe nucleic acid/transition metal enhancer mixture does not contain anyretroviral sequences and is only transiently transcribed. Alternatively,the nucleic acid codes for a polypeptide that may be expressed by thecellular machinery. The polypeptide may be a functional protein and maybe secreted by the muscle cells into the interstitial spaces within thebrain. Therefore, the methods of the present invention may be used tosupplement various proteins present within the host organism by theaddition of the newly transcribed peptide product in a manner asdescribed above.

5.7 Routes of Administration of the Nucleic Acid/Transition MetalEnhancer Solution

[0116] The nucleic acid/transition metal enhancer mixture may be appliedto target tissues and/or cells using any method capable of exposing,either directly or indirectly, nucleic acids into cells. One of skill inthe art will appreciate that references describing routes ofadministration for “naked” (free) nucleic acid delivery are well suitedfor the methods of the present invention. It will be appreciated thatany known representative administration methods may be adapted topractice the methods according to the present invention. In oneembodiment, the nucleic acid/transition metal enhancer mixture may beadministered intramuscularly using methods derived from, for example,Rivera et al., Proc. Natl. Acad. Sci. U.S.A. 96:8657, 1999, and/orMcCluskie et al, Mol. Med. 5:287, 1999. Additionally, the nucleicacid/transition metal enhancer mixture may be administeredintratracheally using methods adopted from those described by Bennett etal., J. Med. Chem. 40:4069, 1997, and/or Meyer et al, Gene. Ther. 2:450,1995. In yet another embodiment, the nucleic acid/transition metalenhancer mixture may be administered intraperitoneally using methodsadopted from those described by McCluskie et al., id., and/or Reimer etal., J. Pharmacol. Exp. Ther. 289:807, 1999. The nucleic acid/transitionmetal enhancer mixture may be also be administered intradermally usingmethods adopted from those described by McCluskie et al., id. and/orWatanabe et al., J. Immunol. 163:1943, 1999. In another embodiment, thenucleic acid/transition metal enhancer mixture may be administeredintravenously using methods adopted from those described by McCluskie etal., id., and/or Wang et al., J. Clin. Invest. 95:1710, 1995. In stillanother embodiment, the nucleic acid/transition metal enhancer mixturemay be administered intraperineally, subcutaneously, sublingually, viathe vaginal wall, by intranasal instillation, intrarectally, ocularly,intraductally, or orally using adaptations of various methods describedin McCluskie et al., id. In yet another embodiment, the nucleicacid/transition metal enhancer mixture may be administered by intranasalinhalation by adaptations of the methods described in McCluskie et al.,id., or Kulkin et al., J. Virol., 71:3138, 1997. The nucleicacid/transition metal enhancer mixture may also be administeredintravaginally using adaptations of methods described by McCluskie etal., id., or Wang et al., Vaccine 15:821, 1997. Additionally, thenucleic acid/transition metal enhancer mixture may be administeredtopically using adaptations of the route of administration described byYu et al., J. Invest. Dermatol. 112:370, 1999.

5.8 Therapeutic Formulations

[0117] In one embodiment, the nucleic acid/transition metal enhancermixture, according to the method of the present invention, may beprepared in unit dosage form provided in ampules, multidose containers,or other pharmaceutically accepted dosage forms. The nucleicacid/transition metal enhancer mixture may be present in such forms assuspensions, solutions, or emulsions in oily or preferably aqueousvehicles. Alternatively, the nucleic acid/transition metal enhancermixture may be lyophilized to form a lyophilized product. Thelyophilized product may be hydrated, at the time of delivery, with asuitable vehicle, such as sterile pyrogen-free water. Both liquid aswell as lyophilized forms that may be reconstituted will compriseagents, preferably buffers, in amounts necessary to suitably adjust thepH of the injected solution. For any parental use, particularly if theformulation is to be administered intravenously, the total concentrationof the solutes should be controlled to make the desirable preparationisotonic or weakly hypertonic. Nonionic materials, such as sugars, arepreferred for adjusting tonicity, and sucrose is particularly preferred.Any of these forms may further comprise suitable formulatory agents,such as starch or sugar, glycerol or saline. The compositions per unitdosage, whether liquid or solid, may contain from 0.1% to 99% nucleicacid.

[0118] The units dosage ampules or multidose containers, in which thenucleic acids are packaged prior to use, may comprise a hermeticallysealed container enclosing an amount of nucleic acid or solutioncontaining a nucleic acid suitable for a pharmaceutically effective dosethereof, or multiples of an effective dose. The polynucleotide ispackaged as a sterile formulation, and the hermetically sealed containeris designed to preserve the sterility of the formulation until use.

[0119] In a preferred embodiment, the container in which the nucleicacid/transition metal enhancer is packaged employs the usage of theknown Good Manufacturing Practice (GMP) compliant protocol and isappropriately labeled in accordance with applicable sections of theFederal Food, Drug, and Cosmetic Act (the “FDCA”; Title 21, UnitedStates Code).

6. EXPERIMENTS 6.1 EXPERIMENTAL METHODS

[0120] The following experiments are intended to provide those ofordinary skill in the art with a disclosure and description of how tocarry out the invention and is not intended to limit the scope of whatthe inventor regards as the invention.

6.1.1 Preparation and Purification of Reporter Genes

[0121] A DNA vector, pCMV.FOX.Luc-2 (FIG. 1), containing the fireflyluciferase reporter gene (LUC) operably linked to human cytomegalovirusmajor immediate early enhancer/promoter was stably transfected intocompetent E. coli XL-1 blue cells (Stratagene, La Jolla, Calif.),cultured in Luria Bertani (LB) medium, and further isolated by alkalinelysis. The plasmid was subsequently passed through an anion exchangeresin (Qiagen, Santa Clarita, Calif.) to yield an endotoxin-reduced,supercoiled plasmid. Once purified, the plasmid is suspended in asolution containing 10 mM Tris-HCl and 1 mM EDTA. The plasmid DNA,pBAT-iMG-2, containing the alpha-1 antitrypsin gene was prepared andpurified using a similar procedure. The plasmid pCMV.FOX.hGH, containingthe human growth hormone gene, was prepared and purified using a similarprocedure.

6.1.2 Preparation of Nucleic Acid Solutions For In Vivo Transfection

[0122] Nucleic acid/transition metal enhancer mixtures were prepared bysequentially adding deionized water or a buffered solution, DNA, and adesired transition metal enhancer to a polystyrene tube with mixing.“Free” (i.e., “Naked”) DNA controls were prepared by sequentially addingDNA to water or a buffered solution

6.1.3 Administration of Anesthesia

[0123] Intramuscular injection of mixtures comprising ketamine (30mg/kg), xylazine (6.0 mg/kg) and aceproamzine (1.0 mg/kg) wereadministrated to all experimental animals.

6.1.4 Intraductal Delivery of Nucleic Acid/transition Metal EnhancerInto the Rat Salivary Gland

[0124] Male Sprague-Dawley rats (260-280 g) were fasted the night priorto treatment. After administration of the anesthesia (see Section6.1.3), both right and left salivary gland ducts (specifically,Wharton's duct) were cannulated with fine polyurethane tubing (i.d.0.005″) and cemented into the desired location. Atropine was thenadministered subcutaneously (0.5 mg/kg) and, after eight minutes, 50 μlof the nucleic acid/transition metal enhancer mixture was injected,infused, instilled or administered at the ductal orifice or directlyinto the duct at any point along its length. See Goldfine, NatureBiotechnology 15:1378, 1997.

[0125] In embodiments in which a liposome/transition metal/nucleic acidmixture was used, 200 μl of the liposome/transition metal/nucleic acidmixture was injected, infused, instilled or administered at the ductalorifice or directly into the duct at any point along its length. SeeGoldfine, Nature Biotechnology 15:1378, 1997. The liposome/transitionmetal/nucleic acid mixture was prepared by sequentially adding anappropriate amount of sterile water, liposome solution (3:1,N,N,N′,N′-tetramethyl-N,N′-bis(2-hydroxyethyl)-2,3-bis(9(z)-octadecenoyloxy)-1,4-butanediaminiumiodide (DOHBD):DOPE), transition metal enhancer, and the plasmid DNApCMV.FOX.hGH to a polypropylene tube.

[0126] Regardless of whether a liposome/transition metal/nucleic acidmixture or a nucleic acid/transition metal enhancer mixture wasdelivered, the tubing and mixture was kept in place for ten additionalminutes after application of the mixture prior to removal. At 48 hourspost atropine administration, the rats were anesthetized byintraperitoneal injection of pentobarbital (50 mg/kg). The right andleft submandibular glands were surgically removed, and the tissues wereassayed for any observable reporter gene (luciferase of hGH) expressionusing the methods described below.

6.1.5 Luciferase Assay

[0127] The presently claimed invention describes a new method using acombination of a transition metal enhancer and a nucleic acid ofinterest for gene delivery. The present invention provides a methodwhich may enhance gene expression by improving the efficiency of genedelivery. This change in gene expression can be quantitated usingvarious assays, such as the luciferase assay. de Wet et al., Molec. CellBiol. 7:725, 1987. In various experiments described herein, the rightand left submandibular glands were removed from the male Sprague-Dawleyrats at 48 hours post administration of the pCMV.FOX.Luc-2 containingsolution. In additional experiments other organs, such as the rat lungs,were used.

[0128] In experiments in which submandibular glands were tested, theamount of luciferase present in the right and left submandibular glandof each male Sprague-Dawley rat was measured independently and treatedas separate, individual experiments (e.g., trials) because the amount ofluciferase expressed by one submandibular gland is independent of theamount of luciferase expressed in the other submandibular gland.Therefore, each submandibular gland was independently lysed in lysisbuffer (1.0 ml buffer per 0.1 g tissue) to create a lysis homogenate.The lysis buffer contained 100 mM K₂PO₄ pH 7.0, 1 mM dithiothreitol, and1% Triton X-100. A 100 μl aliquot of lysis homogenate from eachsubmandibular gland was analyzed for luciferase activity (de Wet et al.,Molec. Cell Biol. 7:725, 1987) using a Monolight 2010 luminometer(Analytical Luminescence Laboratories). Accordingly, luciferase lightemissions from each aliquot of the lysis homogenate were measured over aten second period.

[0129] Activity was expressed as relative light units, valuescollectively representing the assay conditions, luciferaseconcentration, luminometer photomultiplier tube sensitivity andbackground. Using well known techniques, the luciferase light units maybe converted, for example, to picograms of luciferase protein. See,e.g., Felgner et al., U.S. Pat. No. 5,580,899 at Example 13. Allexperiments were performed in duplicate. In experiments usingsubmandibular glands, data from each individual submandibular glandlysis homogenate is reported as a single trial. Results from multipletrials (n=4) are averaged and listed in Tables 1 through 8 below.

6.1.6. Intra-tracheal Delivery of DNA-Containing Products in the MouseLung

[0130] Male BALB/c mice (specific pathogen free, Charles RiverLaboratories; 20-21 g) were used in the transfection experiments.Anesthesia was provided for all invasive procedures; animals wereterminated by intraperitoneal administration of pentobarbital accordingto standard protocols. Neck dissections were performed on anesthetizedmice using a one centimeter incision through the skin of the anteriorneck. Delivery of 150 μl of the nucleic acid/transition metal enhancerwas performed using a half inch thirty gauge needle, inserted 1-3tracheal ring interspaces inferior to the larynx. For comparison, a freenucleic acid solution (150 μl containing 512 μg of DNA) was prepared insterile water and delivered in a similar manner. After injection, thepoint of incision was repaired using staples. The mice were terminatedwithin 48 hours after treatment. A tracheal/lung block was dissected andthen homogenized in chilled lysis buffer comprising 0.1M potassiumphosphate buffer (pH 7.8), 1% Triton X-100, 1 mM dithiothreitol, and 2mM EDTA, and assayed for luciferase activity.

6.1.7. Intraductal Instillation of DNA-Containing Products into the RatPancreas or Liver

[0131] Male Sprague-Dawley rats (weighing 260-280 g) were fasted thenight prior to treatment. Care was taken to ensure that this procedurewas conducted under sterile conditions. After anesthesia (see above) andlaparotomy, the distal end of the bile duct (at the level of theduodenum) was ligated, the proximal end of the pancreas (at the portahepatis level) was temporary blocked by a ligature, a PE-10 tubing wasinserted through an incision of the bile duct near the duodenum. 0.1 mlof the selected nucleic acid/transition metal enhancer mixture wasinjected in a retrograde manner into the duct. Successful injection wasconfirmed by visible swelling of the gland. For administration to theliver, the proximal portion of the bile duct (prior to its entry intopancreatic tissue) was injected. After DNA delivery, a by-pass operationwas done by directly introducing the bile flow to duodenum through theexterior end of the PE-10 tubing. After carefully ensuring the ligatureswere secure, 1 ml of ampicillin (15 mg/ml) was injected into theperitoneal cavity and the incision was closed in one layer, combiningfascia and skin with 3-0 silk suture. The closed incision was washedwith dilute ethanol and the animal was monitored under a heat lamp untilit was fully awake and ambulatory. The mice were terminated 48 hoursafter treatment. The pancreas and liver were removed and individuallyhomogenized in chilled lysis buffer, and assayed for luciferaseactivity.

6.1.8. Human Alpha-1 Antitrypsin Assay

[0132] Polystyrene 96-well plates (Costar #3590) were coated withprimary coating antibody (rabbit polyclonal; Roche #605 002, diluted1:1000, in 1 X carbonate buffer; use 100 /well), and placed inhumidified hybridization tray and incubate overnight in refrigerator (4°C.). The plate was then washed two times with PBS-T (phosphate bufferedsaline +0.5% Tween-20; 200/well), and blocked with PBS-T+1% BSA (200 μL/well) at room temperature for one hour. After three PBS-T washes, thetest samples were added (100 μL /well) and incubated three hours at roomtemperature on a microplate shaker (500 rpm). After five PBS-T washes,the second antibody was added (goat polyclonal; ICN #55236, diluted1:2000 in PBS-T+1% BSA; 100 μL /well), and incubated sixty minutes on amicroplate shaker (500 rpm). The plate was then washed five times withPBS-T and the TMB substrate was added (Dako #S1600; 100 μL /well). Theassay development required twenty minutes, and was monitored with aplatereader set at 650 nm wavelength (Molecular Devices SpectraMax190,using SOFT max v 3.0 software). At this time, a 2N H₂SO₄ stop solution(100 μL/well) was added, and the final readings were taken at 450 nM.

6.1.9. Preparation of Liposome Solutions

[0133] In one embodiment, an appropriate mass of cationic lipid and theneutral lipid dioleoylphosphatidylethanolamine (DOPE) was added assolutions in chloroform to 1.9 mL sample vials to yield the desiredmolar ratio of cationic lipid:DOPE. The chloroform was removed viarotary evaporation at 37° C. The resulting thin lipid films were placedunder vacuum overnight to insure that all traces of solvent have beenremoved. The lipid mixture was resuspended in 1 mL sterile water at 25°C. until the film is hydrated, and then vortex mixed to afford anemulsion. For the in vitro experiments, these emulsions were formulatedas a cationic lipid concentration of 1 mM. For the in vivo experiments,the emulsions were formulated at a cationic lipid concentration of 3 mM.

6.1.10. Cell Culture

[0134] NIH 3T3 cells were obtained from ATCC (CRL 1658), cultured inDulbecco's Modified Eagle's Medium with ten percent calf serum, andplated on standard 24 well tissue culture plates 24 hours prior totransfection. Cells were approximately eighty percent confluent at thetime of transfection.

6.1.11. Transfection of Cultured Cells

[0135] NIH 3T3 cells were plated onto 24 well tissue culture plates asdescribed in section 6.1.10. The growth media was removed via aspirationand the cells were washed once with 0.5 mL PBS / well. The liposome /transition metal / nucleic acid solutions were formed through sequentialaddition of appropriate amounts of DMEM (without serum), the liposomesolution (1:1N,N-[Bis(2-hydroxyethyl)]-N-methyl-N-[2,3-bis(tetradecanoyloxy)propyl]ammonium chloride (DMDHP):DOPE, zinc chloride, and the plasmid DNApCMV.FOX.Luc.2. The amount of liposome solution used depended on thedesired cationic lipid to DNA phosphate ratio. The addition of thesesubstances was followed by thorough vortex mixing and incubation for 15minutes at room temperature. A 200 microliter aliquot of the resultanttransfection complex was added to each well (I microgram DNA/well, n=4)and the cells were incubated for 4 hours, at 37° C. At this time, 500microliters of DMEM+10% calf serum/well was added and the cells culturedfor approximately 48 hours prior to lysis and analysis. The sampletransfections were subsequently repeated a minimum of three times priorto lysis and analysis.

6.1.12 Luciferase Assay Using Cationic Lipid/Transition MetalEnhancer/Nucleic Acid Complexes

[0136] The presently claimed invention describes a new method using acombination of cationic lipid, transition metal enhancer and a nucleicacid of interest for gene delivery. The present invention provides amethod that may enhance gene expression by improving the efficiency ofgene delivery. This change in gene expression can be quantitated usingvarious assays, such as the luciferase assay. de Wet et al., Molec. CellBiol. 7:725, 1987. In in vitro experiments described herein in which acationic lipid / transition metal enhancer / nucleic acid complex wasused, the cells were lysed, using a lysis buffer, 48 hours postadministration of the pCMV.FOX.Luc-2 containing solution. The lysisbuffer contained 100 mM K₂PO₄ pH 7.0, 1 mM dithiothreitol, and 1% TritonX-100. A 25 μl aliquot of the lysate was analyzed for luciferaseactivity (de Wet et al., Molec. Cell Biol. 7:725, 1987) using aMonolight 2010 luminometer (Analytical Luminescence Laboratories).Accordingly, luciferase light emissions from each aliquot of the lysishomogenate were measured over a ten second period. Activity wasexpressed as relative light units, values collectively representing theassay conditions, luciferase concentration, luminometer photomultipliertube sensitivity and background. Using well known techniques, theluciferase light units may be converted, for example, to picograms ofluciferase protein. See, e.g., Feigner et al., U.S. Pat. No. 5,580,899at Example 13. Results from multiple trials (n=4) are averaged andlisted in Table 10.

6.2 EXPERIMENTAL EXAMPLES

[0137] The following examples are provided to illustrate the methods ofthe presently claimed invention.

6.2.1 Example 1. Effect of DNA Dose on Zinc Chloride-MediatedTransfection

[0138] An experiment was performed to determine the optimal DNA dose forin vivo zinc chloride-mediated transfection. To perform this study,DNA/zinc mixtures, A-1 thru A-4, were prepared by mixing an appropriateamount of water, zinc chloride, and pCMV.FOX.Luc.2 plasmid DNA in apolystyrene tube. The relative amount of zinc chloride to DNA wasmaintained at 0.19 mg zinc chloride per 1 mg DNA. For comparison, DNAcontrol solutions, B-1 thru B-4, were prepared in a similar mannerexcept without zinc chloride. Both the DNA/zinc mixtures and the controlsolutions were screened for in vivo transfection activity at DNA dosesof 32, 64, 96, 128 micrograms by using the rat salivary gland model asdescribed above. Specifically, 50 μl of a particular DNA/zinc mixture ora DNA control solution was administered to both the right and the leftsubmandibular gland of four male Sprague-Dawley rats. At 48 hours postadministration, the glands were harvested and assayed for luciferasespecific activity as described above. The average result obtained fromeach treatment condition examined in this study is presented in Table 1.The data illustrates that DNA/zinc mixtures show higher levels oftransfection activity in the rat salivary gland relative to free DNAsolutions. In addition, the data shows that the improved transfectionactivity is observed using several DNA doses and zinc concentrations.TABLE 1 Effect of DNA Dose on Zinc Chloride-Mediated TransfectionRelative DNA Buffer Light Solution Dose [μg] ZnCl₂ [mM] Units A-1 32 0.91.6  57884 A-2 64 1.8 3.2 145179 A-3 96 2.7 4.8 192936 A-4 128  3.6 6.4756838 B-1 32 0 1.6  24322 B-2 64 0 3.2  31885 B-3 96 0 4.8  59774 B-4128  0 6.4  36195

6.2.2 Example 2. Nickel-Mediated In Vivo Transfection

[0139] An experiment was conducted to determine if nickel promotes invivo transfection. To perform this study, DNA/nickel mixtures wereprepared by sequentially adding an appropriate amount of water, nickelchloride, and pCMV.FOX.Luc.2 plasmid DNA to a polystyrene tube withmixing. Mixtures were prepared at 0.3 mM and 0.9 mM nickel chloride andscreened for in vivo transfection activity by administering 50 μl of themixture, containing 32 μg DNA, to the right and left submandibular glandof male Sprague-Dawley rats. For comparison, 50 μl of a DNA/zinc mixture(0.9 mM zinc chloride, and 32 ,μg DNA) was also administered to thesubmandibular glands of rats. At 48 hours post administration, theglands were harvested and assayed for luciferase specific activity asdescribed above. The average results obtained from eight individualglands are presented in Table 2. The results from this study demonstratethat nickel promotes in vivo transfection in a dose dependent manner. Inaddition, nickels ability to promote transfection is similar to thatobserved by using zinc. TABLE 2 Comparison of NiCl₂— and ZnCl₂—MediatedIn Vivo Transfection Metal Concentration Chloride^(†) [mM]^(‡)Average^(§) Ni 0.3 18391 Ni 0.9 65121 Zn 0.9 63842

[0140] † Metal Chloride present in nucleic acid/transition metalenhancer solution

[0141] ‡ Concentration of metal chloride used in nucleic acid/transitionmetal enhancer solution

[0142] § Average relative light units from eight rat submandibularglands

6.2.3 Example 3. Copper-Mediated In Vivo Transfection

[0143] An experiment was conducted to determine if copper promotes invivo transfection. To perform this study, DNA/copper mixtures wereprepared by sequentially adding an appropriate amount of water,Tris-HCl, EDTA, cuprous chloride, and DNA (pCMV.FOX.Luc.2) to apolystyrene tube. Mixtures were prepared at 0.3 mM, 0.9 mM, and 1.2 mMcuprous chloride and screened for in vivo transfection activity byadministering 50 μl of a particular mixture, containing 32 μg DNA, tothe right and left submandibular gland of four male Sprague-Dawley rats.For comparison, 50 μl of a DNA/zinc mixture (0.9 mM zinc chloride, and32 μg DNA) was also administered to the submandibular glands of fourrats. At 48 hours post administration, the glands were harvested andassayed for luciferase specific activity as described above. The averageresult obtained from each treatment condition examined in this study ispresented in Table 3. The results demonstrate that copper can also beused to promote in vivo transfection. In addition, coppers ability topromote transfection is superior to that observed by using zinc. TABLE 3Comparison of CuCl₂— and ZnCl₂—Mediated In Vivo Transfection MetalConcentration Chloride^(†) [mM]^(‡) Average^(§) Cu 0.6 17667 Cu 0.942204 Cu 1.2 17194 Zn 0.9  5685

[0144] † Metal Chloride present in nucleic acid/transition metalenhancer solution

[0145] ‡ Concentration of metal chloride used in nucleic acid/transitionmetal enhancer solution

[0146] § Average relative light units from eight rat submandibularglands

6.2.4 Example 4. Cobalt-Mediated In Vivo Transfection

[0147] An experiment was conducted to determine if cobalt promotes invivo transfection. To perform this study, DNA/cobalt mixtures wereprepared by sequentially adding an appropriate amount of water, cobaltchloride, and DNA (pCMV.FOX.Luc.2) to a polystyrene tube. Mixtures wereprepared at 0.3 mM and 0.9 mM cobalt chloride and screened for in vivotransfection activity by administering 50 μl of a particular mixture,containing 32 μg DNA, to the right and left submandibular gland of fourmale Sprague-Dawley rats. For comparison, 50 μl of a “free” DNA solution(32 μg DNA) was also administered to the submandibular glands of fourrats. At 48 hours post administration, the glands were harvested andassayed for luciferase specific activity as described above. The averageresult obtained from each treatment condition examined in this study ispresented in Table 4. The results from this study demonstrate thatcobalt promotes in vivo transfection when compared to a “free” DNAsolution. In addition, the observed improvement in transfection activitywas observed at both of the cobalts concentrations screened. TABLE 4Effect of CoCl₂ on In Vivo Transfection^(§) [CoCl₂] (mM) Average — 236980.3 37219 0.9 44926

[0148] § Data in each trial represents relative light units producedduring the luciferase assay.

6.2.5 Example 5. Transition Metal-Mediated Transfection of the MouseLung

[0149] An experiment was conducted to determine if transition metalspromote in vivo transfection of the mouse lung. To perform this study, aDNA/zinc mixture was prepared by sequentially adding an appropriateamount of water, zinc chloride, and DNA (pCMV.FOX.Luc.2) to apolystyrene tube. The final zinc chloride concentration of this mixturewas 3.6 mM. The mixtures was screened for in vivo transfection activityby administering 150 μl of the mixture, containing 384 μg DNA,intratracheally to lungs of four male BALB/c mice as described above.For comparison, 150 μl of a DNA solution (354 μg DNA) was alsoadministered to intratracheally to the lungs of four mice. At 48 hourspost administration, the tracheal/lung block was dissected and assayedfor luciferase specific activity as described above. The average resultobtained from each treatment condition examined in this study ispresented in Table 5. The results from this study demonstrate thattransition metals can be used to promote in vivo transfection of themouse lung. In this particular case, zinc chloride improves observedtransfection activity by 5-fold relative to the “free” DNA solution.TABLE 5 Effect of ZnCl₂ on Transfection of the Mouse Luna^(§) TreatmentCondition Average “free” DNA  535.2 DNA + ZnCl₂ 2715.8

[0150] § Data in each trial represents relative light units producedduring the luciferase assay.

6.2.6 Example 6. Influence of Metal Ligand Substitution on TransitionMetal-Mediated Transfection

[0151] An experiment was conducted to determine if transition metalcompounds other than transition metal chlorides have the ability topromote in vivo transfection. In this study, zinc chloride was comparedto zinc sulfate and zinc acetate for the ability to enhance transfectionof the rat salivary gland. For each zinc-containing compound, DNA/zincmixtures were prepared by sequentially adding an appropriate amount ofwater; the zinc containing compound, either zinc chloride, zinc acetate,or zinc sulfate; and DNA (pCMV.FOX.Luc.2) to a polystyrene tube. Thefinal zinc concentration of each mixture was 3.6 mM. The relativetransfection activity of each zinc compound was determined byadministering 50 μl of the DNA/zinc mixture (containing 128 μg DNA) intothe right and left submandibular gland of male Sprague-Dawley rats. At48 hours post administration, the glands were harvested and assayed forluciferase specific activity as described above. The average resultobtained from each treatment condition examined in this study ispresented in Table 6. The results demonstrate that zinc sulfate and zincacetate are better than zinc chloride at promoting in vivo transfection.The study also demonstrates that transition metal compounds containingeither organic ligands (acetate) or inorganic ligands (sulfate andchloride) are capable of promoting in vivo transfection. TABLE 6 Effectof Zinc Ligand Structure on Observed Transfection Activity in the RatSaliva Gland^(§) TransitionMetal Enhancer Average Zn(CH₃CO₂)₂ 309965ZnCl₂ 243362 ZnSO₄ 355676

[0152] § Data in each trial represents relative light units producedduring the luciferase assay.

6.2.7 Example 7. Influence of pH on Transition Metal-MediatedTransfection

[0153] An experiment was conducted to determine the effect pH has ontransition metal-mediated in vivo transfection. DNA/zinc mixturescontaining 3.6 mM zinc chloride were prepared at pH 5.5, 6.5, 7.5 and8.5. The relative transfection activity of these DNA/zinc mixtures wasdetermined by administering 50 μl of each DNA/zinc mixture (containing128 μg DNA) into the right and left submandibular glands of maleSprague-Dawley rats. For comparison, four rats received injections of a“free DNA” solution (50 μl, 128 μg, pH 7.5). At 48 hours postadministration, the glands were harvested and assayed for luciferasespecific activity as described above. The average result (n=4) obtainedfor each treatment condition examined in this study is presented inTable 7. The results demonstrate that, for the solutions screened, thepH of the zinc/DNA solution has a negligible effect on observedtransfection activity. TABLE 7 Effect of pH on ZnCl₂-MediatedTransfection of the Rat Salivay Gland^(§) [ZnCl₂] (mM) pH Average 3.65.5 252446 3.6 6.5 196002 — 7.5  52397 3.6 7.5 260340 3.6 8.5 277958

[0154] § Data in each trial represents relative light units producedduring the luciferase assay.

6.2.8 Example 8. Influence of Media Composition on TransitionMetal-Mediated Transfection

[0155] An experiment was conducted to determine if Tris-HCl and EDTA areessential components for an active nucleic acid/transition metalenhancer mixture. Tris-HCl and EDTA are commonly used as preservativesfor DNA solutions. Tris-HCl and EDTA, collectively referred to as TE,inhibit DNase activity therefore preventing enzymatic degradation of DNAsolutions. EDTA binds to calcium and magnesium ions, which are requiredfor DNase activity. EDTA is also known to have an affinity for zinc andother transition metals. Since all the experiments mentioned above usednucleic acid/transition metal enhancer mixtures containing Tris-HCl andEDTA, the influence of these additives was studied. A test set ofnucleic acid/transition metal enhancer mixtures, C-1 thru C-4, wereprepared each differing in the concentrations of EDTA and Tris-HClcontained within them (See Table 8 for the composition of eachsolution). A set of control mixtures, D-1 thru D-4, corresponding tosolutions C-1 thru C-4, was also prepared (See Table 8 for thecomposition of each solution). The control set of mixtures, D-1 thruD-4, were identical to the test mixtures, C-1 thru C-4, except that nozinc chloride was present in the control set. These mixtures werescreened for transfection activity using the rat salivary gland model.At 48 hours post administration, the glands were harvested and assayedfor luciferase specific activity as described above. The average result(n=4) obtained from each treatment condition examined in this study ispresented in Table 8. The results of this study indicate that Tris-HCland EDTA are not important components for an active DNA/zinctransfection mixture. However, DNA/zinc mixtures containing Tris-HCl andEDTA are more active than a “free” DNA solution (Table 1). Resultsobtained from this experiment suggest that “active” DNA/zinc mixturesmay be prepared using many different formulation conditions. TABLE 8Influence of Transfection Media Composition on ZnCl₂—MediatedTransfection^(§) [ZnCl₂] [Tris-HCl] [EDTA] Relative Light Solution (mM)(mM) (mM) Units C-1 3.6 10 1 437597  C-2 3.6 10 0 139291  C-3 3.6  0 1414083  C-4 3.6  0 0 1354218  D-1 0 10 1 26145 D-2 0 10 0 30790 D-3 0  01 38999 D-4 0  0 0 42413

6.2.9 Example 9. Zinc-Mediated Transfection of Rat Salivary Gland withAlpha-1 Antitrypsin.

[0156] An experiment was conducted to determine if transitionmetal-mediated transfection could be used to introduce an alpha-1antitrypsin gene into the cells of the rat salivary gland. Alpha-1antitrypsin is a secreted protein found in blood. In order to performthis study, a plasmid DNA containing the alpha-1 antitrypsin gene wasprepared using procedures similar to those used to prepare theluciferase plasmid. A DNA/zinc mixture was prepared by sequentiallyadding an appropriate amount of water, zinc chloride, and DNA(pBAT-iMG-2) to a polystyrene tube. Aliquots of this mixture, 50 μlcontaining 128 mg DNA, were then injected into both the right and leftsubmandibular glands of four rats. For comparison, 50 ml of a “free” DNAsolution (128 mg DNA) were also administered to the submandibular glandsof four rats. At 48 hours post administration, the glands wereharvested, homogenized in lysis buffer (100 mM K₂PO₄, pH 7.0, 1 mMdithiothreitol, and 1% Triton X-100), then assayed for the presence ofalpha-1 antitrypsin using the method described above. The results listedin Table 9 show that administration of a DNA/zinc mixture leads tohigher levels of alpha-1 antitrypsin expression than administration of a“free” DNA solution. TABLE 9 Effect of ZnCl₂ on Observeda-1-anti-Trypsin Expression in the Rat Salivary Gland α-1-AT Expression[ZnCl₂] (mg/mL) — 19.4 3.6 31.6

6.2.10 Example 10. Effect of ZnCl₂ on Observed Luciferase Expression inthe Rat Pancreas

[0157] Experiments were performed to determine the effect of ZnCl₂ onluciferase expression using the rat pancreas model described in section6.1.7. Using the luciferase assay as described above, the relativeeffectiveness of pCMV.FOX.Luc-2 (i) in the absence of ZnCl₂ and (ii) inthe presence of ZnCl₂ were each tested in independent trials. In eachtrial, 64 μg of pCMV.FOX.Luc-2 in a total volume of 100 μL was injectedinto the bile duct near the duodenum as described in section 6.1.7. Intrials conducted in the presence of ZnCl₂, the concentration of ZnCl₂ inthe injected solution was 1.8 mM. Luciferase activity was assayed after48 hours of treatment. The average luciferase activity from the fourtrials conducted in the absence of ZnCl2 was 7274 relative luciferaselight units per 10 mg of pancreatic tissue. In contrast, the averageluciferase activity from the four trials in which ZnCl₂ was 22028relative luciferase light units per 10 mg of pancreatic tissue. Theexperiments demonstrate that the presence of ZnCl₂ significantlyenhanced luciferase expression in the rat pancreas.

6.2.11 Example 11. Effect of Added Zinc on Cationic Liposome-MediatedGene Delivery to NIH 3T3 Cells

[0158] An experiment was performed to demonstrate that ZnCl₂ can be usedto enhance the in vitro transfection activity of cationic lipid/nucleicacid complexes. To perform this study, cationic lipid/nucleic acid/zincmixtures were prepared by mixing appropriate amounts of serum-free DMEM,cationic liposomes, zinc chloride and pCMV.FOX.Luc.2 plasmid DNA in apolystyrene tube. In this experiment, the cationic lipid/nucleic acidcomplexes were formed at different cationic lipid:nucleic acid phosphatecharge ratios. Specifically, complexes were formed at charge ratios of0.5, 0.75, 1.0, and 2.0. Complexes formed at charge ratios above 1.0possess a net positive charge whereas those with a charge ratio below1.0 have a net negative charge. The cationic lipid/nucleic acidphosphate charge ratio is an important experimental parameter thatinfluences the transfection activity of cationic lipid/nucleic acidcomplexes. In many instances, complexes possessing a net positive chargeare more active than those with a net neutral or net negative charge.Cationic lipid/nucleic acid complexes at each charge ratio were screenedfor transfection activity in NIH 3T3 cells in the presence of differentconcentrations of zinc chloride (0.0, 0.1, 1, 10, 100, and 1000 μM). NIH3T3 cells are a murine fibroblast tissue culture cell line commonly usedto demonstrate the in vitro transfection activity of gene deliveryreagents. After 48 hours post-application of the cationic lipid/DNA/zincsolutions to the cells, the cells were lysed with lysis buffer and thelysate was assayed for luciferase specific activity. As illustrated inFIG. 3 and Table 10, the data clearly illustrates that zinc, when addedto a cationic liposome/DNA mixture, can enhance in vitro transfection ofcultured NIH 3T3 murine fibroblast cells by two to forty fold dependingon the cationic lipid to nucleic acid charge ratio. The effect was morepronounced at lower charge ratios, however, and effect was observed atall the charge ratios screened. The ability of the methods of thepresent invention to increase the activity of low charge ratio cationiclipid/DNA complexes is highly advantageous over prior art systemsbecause highly charged complexes have a significant amount of associatedcytotoxicity. TABLE 10 Effect of Added Zinc on CationicLiposome-Mediated Gene Delivery to NIH 3T3 Cells Lipid/DNA Charge RatioZinc Concentration (μM) 0 0.1 1 10 100 1000 0.5 34366.5 357320 289649.8296498.3 690512.3 44053.75 0.75 113887 528118.8 141950 397689.3 334007.5298625.5 1 869865 931446.8 835699.8 1366067 359188.5 414392 2 767710677663.5 747069.5 1215879 827479.8 109543.8 ZnCl₂ (μM) Trial 1 Trial 2Trial 3 Trial 4 Average 0.5 Charge Ratio 0 3146 61387 21468 5146534366.5 0.1 487868 426369 256897 258146 357320 1 385049 200471 304108268971 289649.8 10 325001 323001 250110 287881 296498.3 100 351939252969 294018 1863123 690512.3 1000 43835 40236 59395 32749 44053.750.75 Charge Ratio 0 118667 128866 103034 104981 113887 0.1 613179 556656437658 504982 528118.8 1 127339 236302 123913 80246 141950 10 455299465986 362168 307304 397689.3 100 404983 351094 358591 221362 334007.51000 335343 257292 374511 227356 298625.5 1 Charge Ratio 0 617205 8762291077503 908523 869865 0.1 901184 862221 1113520 848862 931446.8 1 942667864670 799992 735470 835699.8 10 2145687 1433861 1202748 681970 1366067100 262768 379927 447492 346567 359188.5 1000 437998 443459 502263273848 414392 2 Charge Ratio 0 1017767 777548 684552 590973 767710 0.1596911 685311 818392 610040 677663.5 1 661803 814409 754168 757898747069.5 10 1498720 1238992 1207690 918114 1215879 100 768327 880621941720 719251 827479.8 1000 199493 48657 189902 123 109543.8

6.2.12 Example 12. Effect of Added Zinc on Cationic Liposome-MediatedGene Delivery to Rat Submandibular Gland

[0159] An experiment was performed to demonstrate that zinc chloride canbe used to enhance the in vivo transfection activity of cationiclipid/nucleic acid complexes. To perform this study, cationiclipid/nucleic acid/zinc mixtures were prepared by mixing the appropriateamount of sterile water, cationic liposomes, zinc chloride andpCMV.FOX.hGH plasmid DNA in a polystyrene tube. Specifically, 40microliters of a 3 mM 3:1 DOHBD:DOPE liposome mixture, 270 microlitersof a 8.08 microgram / microliter pCMV.FOX.hGH plasmid DNA mixture, andan appropriate amount of a 70 mM zinc chloride mixture were added to apolystyrene tube containing a sufficient amount of water to give a finaltotal volume of 2500 microliters. In this experiment, the final zincchloride concentration was either 0.125 mM, 0.250 mM, or no zinc at all.The cationic lipid:DNA phosphate ratio in this study remained constantfor all mixtures screened. Once prepared, 200 microliters of eachmixture was instilled into the right and left submandibular glands offour male Sprague-Dawley rats. Thus, 175 micrograms of the plasmid DNAwas instilled into each gland. After one week post-administration of thecationic lipid/nucleic acid/zinc mixtures, the salivary gland of therats were extracted, mixed with a phosphate lysis buffer (10 mM, pH 8.0)and homogenized. The homogenate was then assayed for human growthhormone protein expression. The data (Table 11) illustrates that zinc,when added to a cationic liposome/nucleic acid mixture, can enhance invivo transfection of the rat submandibular gland by at least two foldwhen compared to a cationic liposome/nucleic acid mixture not containingzinc. TABLE 11 Effect of Added Zinc on Cationic Liposome-Mediated GeneDelivery to Rat Submandibular Gland Trial Trial Trial Trial Trial TrialTrial Trial [Zn] mM 1 2 3 4 5 6 7 8 0 292.9 287.9 285.2 255.2 128.6 86.4335.6 277.5 0.125 452.1 360.8 261.7 355.7 517.2 575.6 943.5 1084 0.25857.731 753.3 272.6 315.3 604.3 449.9

[0160] [Zn] mM Average Standard Deviation 0 216.5889 87.67482 0.125505.6361 294.0226 0.25 464.7687 236.9247

References Cited

[0161] All references cited are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes. Many modifications and variations of thisinvention can be made without departing from its spirit and scope, aswill be apparent to those skilled in the art. For example, it is to beunderstood that the invention is not limited to the particularmethodology, protocols, cell types, tissues, vectors and reagentsdescribed because they may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled.

We claim:
 1. A method for delivering a DNA to a mammal, said DNAencoding a peptide or protein operably linked to a promoter, the methodcomprising exposing said mammal to a composition comprising an ionizableor ionized transition metal enhancer and said DNA, wherein said DNAexpresses said peptide or protein.
 2. The method of claim 1 , whereinsaid DNA is expressed in a secretory gland of said mammal.
 3. The methodof claim 2 , wherein the secretory gland is selected from the groupconsisting of a salivary gland, a pancreas, a mammary gland, a thyroid,a thymus, a pituitary gland, and a liver.
 4. The method of claim 3 ,wherein the secretory gland is a salivary gland or a pancreas.
 5. Themethod of claim 2 , wherein said peptide or protein is secreted orreleased from said secretory gland.
 6. The method of claim 2 , whereinthe peptide or protein is not secreted from the secretory gland.
 7. Themethod of claim 1 , wherein said DNA is expressed in the lung, muscle,brain, blood, breast, bone, bladder, skin, liver, stomach, intestine,kidney, testes, uterus, testes, uterus, gastrointestinal tract, orovaries of said mammal.
 8. The method of claim 7 , wherein said DNA isexpressed in the lung, muscle, or brain of said mammal.
 9. The method ofclaim 8 , wherein said DNA is expressed in the lung of said mammal. 10.The method of claim 1 , wherein said composition is delivered to saidmammal by a route of administration selected from the group consistingof intramuscular, intratracheal, intraperitoneal, intradermal,intravenous, intraperineal, subcutaneous, sublingual, intranasalinhalation, intranasal instillation, intrarectal, intravaginal, ocular,oral, intraductal, and topical administration.
 11. The method of claim 1, wherein the composition is a solution having a pH of about 4.0 toabout 9.0.
 12. The method of claim 11 , wherein the composition is asolution having a pH of about 5.5 to about 8.5.
 13. The method of claim1 , wherein the composition is a solution having a total saltconcentration of less than about 250 micromolar.
 14. The method of claim1 , wherein the composition is a solution having a cumulative saltconcentration of less than about 50 micromolar.
 15. The method of claim1 , wherein said mammal is exposed to about 1 microgram to about 100milligrams of the DNA.
 16. The method of claim 1 , wherein said mammalis exposed to about 30 micrograms to about 30 milligrams of the DNA. 17.The method of claim 1 , wherein a molar ratio of the ionizable orionized transition metal enhancer to DNA in the composition is about0.0001:1 to about 1:0.0001.
 18. The method of claim 1 , wherein theionizable or ionized transition metal enhancer is a complex, adduct,cluster or salt of an element selected from the group consisting of ad-block element, a first row ƒ-block element, aluminum, and gallium. 19.The method of claim 18 , wherein the ionizable or ionized transitionmetal enhancer is a complex, adduct, cluster or salt of an elementselected from the group consisting of zinc, nickel, cobalt, copper,aluminum, and gallium.
 20. The method of claim 19 , wherein theionizable or ionized transition metal enhancer is selected from thegroup consisting of zinc sulfate, zinc acetate, nickel sulfate, nickelacetate, cobalt sulfate, cobalt acetate, copper sulfate, and copperacetate.
 21. The method of claim 20 , wherein the ionizable or ionizedtransition metal enhancer is zinc acetate or zinc sulfate.
 22. Themethod of claim 1 , wherein the composition is a solution and theionizable or ionized transition metal enhancer is about 0.01 millimolarZnCl₂ to about 250 millimolar ZnCl₂ in said solution.
 23. The method ofclaim 22 , wherein the ionizable or ionized transition metal enhancer isabout 0.03 millimolar zinc sulfate to about 6.0 millimolar zinc sulfatein said solution.
 24. The method of claim 1 , wherein the composition isa solution and the ionizable or ionized transition metal enhancer isabout 0.01 millimolar zinc acetate to about 250 millimolar zinc acetatein said solution.
 25. The method of claim 1 , wherein the composition isa solution and the ionizable or ionized transition metal enhancer isabout 0.03 millimolar zinc acetate to about 6.0 millimolar zinc acetatein said solution.
 26. The method of claim 1 , wherein the ionizable orionized transition metal enhancer is selected from the group consistingof zinc halide, nickel halide, cobalt halide, copper halide, aluminumhalide, and gallium halide.
 27. The method of claim 26 , wherein theionizable or ionized transition metal enhancer is selected from thegroup consisting of ZnCl₂, NiCl₂, CoCl₂, CuCl₂, AlCl₂, and GaCl₂. 28.The method of claim 1 , wherein the composition is a solution and theionizable or ionized transition metal enhancer is about 0.01 millimolarZnCl₂ to about 250 millimolar ZnCl₂ in said solution.
 29. The method ofclaim 1 , wherein the composition is a solution and the ionizable orionized transition metal enhancer is about 0.03 millimolar ZnCl₂ toabout 6.0 millimolar ZnCl2 in said solution.
 30. The method of claim 1 ,wherein the composition is a solution and the ionizable or ionizedtransition metal enhancer is about 0.01 millimolar NiCl₂ to about 250millimolar NiCl₂ in said solution.
 31. The method of claim 30 , whereinthe ionizable or ionized transition metal enhancer is about 0.03millimolar NiCl₂ to about 6.0 millimolar NiCl₂ in said solution.
 32. Themethod of claim 1 , wherein the composition is a solution and theionizable or ionized transition metal enhancer is about 0.01 millimolarCoCl₂ to about 250 millimolar CoCl₂ in said solution.
 33. The method ofclaim 32 , the ionizable or ionized transition metal enhancer is about0.03 millimolar CoCl₂ to about 6.0 millimolar CoCl₂ in said solution.34. The method of claim 1 , wherein the composition is a solution andthe ionizable or ionized transition metal enhancer is about 0.01millimolar CuCl₂ to about 250 millimolar CuCl2 in said solution.
 35. Themethod of claim 34 , wherein the ionizable or ionized transition metalenhancer is about 0.03 millimolar CuCl₂ to about 6.0 millimolar CuCl₂ insaid solution.
 36. The method of claim 1 , wherein the composition is asolution and the ionizable or ionized transition metal enhancer is about0.01 millimolar AlCl₂ to about 250 millimolar AlCl₂ in said solution.37. The method of claim 36 , wherein the ionizable or ionized transitionmetal enhancer is about 0.01 millimolar AlCl₂ to about 250 millimolarAlCl₂ in said solution.
 38. The method of claim 1 , wherein the DNA is aplasmid.
 39. The method of claim 1 , wherein said protein is selectedfrom the group consisting of insulin, human growth hormone,erythopoietin, clotting factor VII, bovine growth hormone, plateletderived growth factor, clotting factor VIII, thrombopoietin,interleukin- 1, interluekin-2, interleukin-1 RA, superoxide dismutase,catalase, fibroblast growth factor, neurite growth factor, granulocytecolony stimulating factor, L-asparaginase, uricase, chymotrypsin,carboxypeptidase, sucrase, calcitonin, Ob gene product, glucagon,transforming growth factor, ciliary neurite transforming factor,insulin-like growth factor-1, granulocyte macrophage colony stimulatingfactor, interferon (α2A, brain-derived neurite factor, insulintropin,tissue plasminogen activator, urokinase, streptokinase, adenosinedeamidase, calcitonin, arginase, phenylaline ammonia lyase,γ-interferon, pepsin, trypsin, elastase, lactase, intrinsic factor,cholecystokinin, insulinotrophic hormone clotting factor I,glucagon-like protein-I, a-1-antitrypsin, glucocerebrosidase, cysticfibrosis transreductase, angiostatin, endostatin, angiogenics, andantiangiogenics.
 40. A method of delivering a DNA into a mammalianpancreas, liver, salivary gland or mouse lung, said DNA encoding apeptide or protein operably linked to a promoter, the method comprisingexposing said mammal to a composition comprising said DNA and anionizable or ionized transition metal enhancer selected from the groupconsisting of zinc chloride, copper chloride, nickel chloride, cobaltchloride, zinc sulfate, and zinc acetate, wherein said DNA is expressed.41. A method for delivering a DNA to a cell of a mammal, said DNAencoding a peptide or protein operably linked to a promoter, the methodcomprising administering into said cell of said mammal a compositioncomprising an ionizable or ionized transition metal enhancer and saidDNA.
 42. The method of claim 1 , said composition further comprising ana cationic lipid.
 43. The method of claim 42 , wherein a cationiclipid:DNA phosphate ratio of said composition is about 0.01 to about 12.44. The method of claim 42 , wherein said cationic lipid is selectedfrom the group consisting of 1:1N,N-[bis(2-hydroxyethyl)]-N-methyl-N-[2,3-bis(tetradecanoyloxy)propyl]ammoniumchloride andN,N,N′N′-tetramethyl-N,N′-bis(2-hydroxyethyl)-2,3-bis(9(z)-octadecenoyloxy)-1,4-butanediaminiumiodide.